Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Nobuaki Matsumori Last modified date:2019.05.14

Professor / Inorganic Chemistry and Analytical Chemistry / Department of Chemistry / Faculty of Sciences


Papers
1. Masataka Inada, Masanao Kinoshita, Ayumi Sumino, Shigetoshi Oiki, Nobuaki Matsumori, A concise method for quantitative analysis of interactions between lipids and membrane proteins, Analytica Chimica Acta, 10.1016/j.aca.2019.01.042, 1059, 103-112, 2019.06, Although interactions between lipids and membrane proteins (MPs) have been considered crucially important for understanding the functions of lipids, lack of useful and convincing experimental methods has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance (SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip surface with self-assembled monolayer (SAM) with C
6
-chain. To develop this method, we used bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits of using C
6
-SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM provides immobilized MPs with a partial membranous environment, which is important for the stabilization of MPs; and (3) a thinner C
6
-SAM layer (1 nm) compared with MP size forces the MP to bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C
6
-SAM is 10 times higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation, demonstrating the validity and utility of this method..
2. Takaaki Matsufuji, Masanao Kinoshita, Nobuaki Matsumori, Preparation and Membrane Distribution of Fluorescent Derivatives of Ceramide, Langmuir, 10.1021/acs.langmuir.8b03176, 2019.01, Ceramide is a bioactive lipid with significant roles in several biological processes including cell proliferation, apoptosis, and raft formation. Although fluorescent derivatives of ceramide are required to probe the behavior of ceramide in cells and cell membranes, commercial fluorescent ceramide derivatives do not reproduce the membrane behavior of native ceramide because of the introduction of bulky fluorophores in the acyl chain. Recently, we developed novel fluorescent analogs of sphingomyelin in which the hydrophilic fluorophores, ATTO488 and ATTO594, are attached to the polar head of sphingomyelin via a nonaethylene glycol linker and demonstrated that their partition and dynamic behaviors in bilayer membranes are similar to native sphingomyelin. In this report, by extending the concept used for the development of fluorescent analogs of sphingomyelin, we prepared novel fluorescent ceramides that exhibit membrane behaviors similar to native ceramide and succeeded in visualizing ceramide-rich membrane domains segregated from ceramide-poor domains..
3. Kimberly Cornelio, Rafael Atillo Espiritu, Shinya Hanashima, Yasuto Todokoro, Raymond Malabed, Masanao Kinoshita, Nobuaki Matsumori, Michio Murata, Shinichi Nishimura, Hideaki Kakeya, Minoru Yoshida, Shigeki Matsunaga, Theonellamide A, a marine-sponge-derived bicyclic peptide, binds to cholesterol in aqueous DMSO
Solution NMR-based analysis of peptide-sterol interactions using hydroxylated sterol, Biochimica et Biophysica Acta - Biomembranes, 10.1016/j.bbamem.2018.07.010, 1861, 1, 228-235, 2019.01, Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. The inclusion of cholesterol (Chol) or ergosterol in the phosphatidylcholine membrane is known to significantly enhance the membrane affinity for theonellamide A (TNM-A). We have previously revealed that TNM-A stays in a monomeric form in dimethylsulfoxide (DMSO) solvent systems, whereas the peptide forms oligomers in aqueous media. In this study, we utilized 1H NMR chemical shift changes (Δδ1H) in aqueous DMSO solution to evaluate the TNM-A/sterol interaction. Because Chol does not dissolve well in this solvent, we used 25-hydroxycholesterol (25-HC) instead, which turned out to interact with membrane-bound TNM-A in a very similar way to that of Chol. We determined the dissociation constant, KD, by NMR titration experiments and measured the chemical shift changes of TNM-A induced by 25-HC binding in the DMSO solution. Significant changes were observed for several amino acid residues in a certain area of the molecule. The results from the solution NMR experiments, together with previous findings, suggest that the TNM-Chol complex, where the hydrophobic cavity of TNM probably incorporates Chol, becomes less polar by Chol interaction, resulting in a greater accumulation of the peptide in membrane. The deeper penetration of TNM-A into the membrane interior enhances membrane disruption. We also demonstrated that hydroxylated sterols, such as 25-HC that has higher solubility in most NMR solvents than Chol, act as a versatile substitute for sterol and could be used in 1H NMR-based studies of sterol-binding peptides..
4. Anna Möuts, Elina Vattulainen, Takaaki Matsufuji, Masanao Kinoshita, Nobuaki Matsumori, J. Peter Slotte, On the Importance of the C(1)-OH and C(3)-OH Functional Groups of the Long-Chain Base of Ceramide for Interlipid Interaction and Lateral Segregation into Ceramide-Rich Domains, Langmuir, 10.1021/acs.langmuir.8b03237, 34, 51, 15864-15870, 2018.12, Ceramides are important intermediates in sphingolipid biosynthesis (and degradation) and are normally present in only small amounts in unstressed cells. However, following the receptor-mediated activation of neutral sphingomyelinase, sphingomyelin can acutely give rise to substantial amounts of ceramides, which dramatically alter membrane properties. In this study, we have examined the role of the 1-OH and 3-OH functional groups of ceramide for its membrane properties. We have specifically examined how the oxidation of the primary alcohol to COOH or COOMe in palmitoyl ceramide (PCer) or the removal of either the primary alcohol or C(3)-OH (deoxy analogs) affected ceramides' interlipid interactions in fluid phosphatidylcholine bilayers. Measuring the time-resolved fluorescence emission of trans-parinaric acid, or its steady-state anisotropy, we have obtained information about the propensity of the ceramide analogs to form ceramide-rich domains and the thermostability of the formed domains. We observed that the oxidation of the primary alcohol to COOH shifted the ceramide's gel-phase onset concentration to slightly higher values in 1-palmitoyl-2-oleoyl-sn-3-glycero-3-phosphocholine (POPC) bilayers. Methylation of the COOH function of the ceramide did not change the segregation tendency further. The complete removal of the primary alcohol dramatically reduced the ability of 1-deoxy-PCer to form ceramide-rich ordered domains. However, the removal 3-OH (in 3-deoxy-PCer) had only small effects on the lateral segregation of the ceramide analog. The thermostability of the ceramide-rich domains in the POPC bilayers decreased in the following order: 1-OH > COOH > COOMe = 3-deoxy > 1-deoxy. We conclude that ceramide needs a hydrogen-bonding-competent functional group in the C(1) position to be able to form laterally segregated ceramide-rich domains of high packing density in POPC bilayers. The presence or absence of 3-OH was not functionally critical for ceramide's lateral segregation properties..
5. Yo Yano, Shinya Hanashima, Tomokazu Yasuda, Hiroshi Tsuchikawa, Nobuaki Matsumori, Masanao Kinoshita, Md Abdullah Al Sazzad, J. Peter Slotte, Michio Murata, Sphingomyelin Stereoisomers Reveal That Homophilic Interactions Cause Nanodomain Formation, Biophysical Journal, 10.1016/j.bpj.2018.08.042, 115, 8, 1530-1540, 2018.10, Sphingomyelin is an abundant lipid in some cellular membrane domains, such as lipid rafts. Hydrogen bonding and hydrophobic interactions of the lipid with surrounding components such as neighboring sphingomyelin and cholesterol (Cho) are widely considered to stabilize the raft-like liquid-ordered (Lo) domains in membrane bilayers. However, details of their interactions responsible for the formation of Lo domains remain largely unknown. In this study, the enantiomer of stearoyl sphingomyelin (ent-SSM) was prepared, and its physicochemical properties were compared with the natural SSM and the diastereomer of SSM to examine possible stereoselective lipid-lipid interactions. Interestingly, differential scanning calorimetry experiments demonstrated that palmitoyl sphingomyelin, with natural stereochemistry, exhibited higher miscibility with SSM bilayers than with ent-SSM bilayers, indicating that the homophilic sphingomyelin interactions occurred in a stereoselective manner. Solid-state 2H NMR revealed that Cho elicited its ordering effect very similarly on SSM and ent-SSM (and even on the diastereomer of SSM), suggesting that SSM-Cho interactions are not significantly affected by stereospecific hydrogen bonding. SSM and ent-SSM formed gel-like domains with very similar lateral packing in SSM/Cho/palmitoyloleoyl phosphatidylcholine membranes, as shown by fluorescence lifetime experiments. This observation can be explained by a homophilic hydrogen-bond network, which was largely responsible for the formation of gel-like nanodomains of SSMs (or ent-SSM). Our previous study revealed that Cho-poor gel-like domains contributed significantly to the formation of an Lo phase in sphingomyelin/Cho membranes. The results of the study presented here further show that SSM-SSM interactions occur near the headgroup region, whereas hydrophobic SSM-Cho interactions appeared important in the bilayer interior for Lo domain formation. The homophilic interactions of sphingomyelins could be mainly responsible for the formation of the domains of nanometer size, which may correspond to the small sphingomyelin/Cho-based rafts that temporally occur in biological membranes..
6. Masanao Kinoshita, Kenichi G.N. Suzuki, Michio Murata, Nobuaki Matsumori, Evidence of lipid rafts based on the partition and dynamic behavior of sphingomyelins, Chemistry and Physics of Lipids, 10.1016/j.chemphyslip.2018.07.002, 215, 84-95, 2018.09, Sphingomyelin (SM)-rich membrane nano-domains, called lipid rafts, have attracted the interest of researchers due to their potential involvement in the formation of signaling platform. Although there are many studies on lipid rafts, the direct observation of lipid rafts is still challenging owing to two critical reasons. One is the lack of an appropriate fluorescent probe mimicking the native behavior of raft lipids; fluorescent labeling often alters the intrinsic disposition of raft lipids. The other is their spatio-temporal stability; the size of lipid rafts is much smaller than the optical resolution of usual microscopy and their lifetime is much shorter than image acquisition duration. These issues are hampering the visualization of lipid rafts. Our review highlights the recent advances in microscopic techniques to visualize the partition and dynamic behavior of SMs, disclosing the detailed structure of lipid rafts. Moreover, we will elucidate the importance of SM-SM interactions in the stabilization of signaling platforms as lipid rafts..
7. Tomohiro Watanabe, Hajime Shibata, Makoto Ebine, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata, Manabu Yoshida, Masaaki Morisawa, Shu Lin, Kosei Yamauchi, Ken Sakai, Tohru Oishi, Synthesis and complete structure determination of a sperm-activating and -attracting factor isolated from the ascidian ascidia sydneiensis, Journal of Natural Products, 10.1021/acs.jnatprod.7b01052, 81, 4, 985-997, 2018.04, For the complete structure elucidation of an endogenous sperm-activating and -attracting factor isolated from eggs of the ascidian Ascidia sydneiensis (Assydn-SAAF), its two possible diastereomers with respect to C-25 were synthesized. Starting from ergosterol, the characteristic steroid backbone was constructed by using an intramolecular pinacol coupling reaction and stereoselective reduction of a hydroxy ketone as key steps, and the side chain was introduced by Julia-Kocienski olefination. Comparison of the NMR data of the two diastereomers with those of the natural product led to the elucidation of the absolute configuration as 25S; thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved..
8. Akira Naito, Nobuaki Matsumori, Ayyalusamy Ramamoorthy, Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy, Biochimica et Biophysica Acta - General Subjects, 10.1016/j.bbagen.2017.06.004, 1862, 2, 307-323, 2018.02, A variety of biomolecules acting on the cell membrane folds into a biologically active structure in the membrane environment. It is, therefore, important to determine the structures and dynamics of such biomolecules in a membrane environment. While several biophysical techniques are used to obtain low-resolution information, solid-state NMR spectroscopy is one of the most powerful means for determining the structure and dynamics of membrane bound biomolecules such as antibacterial biomolecules and amyloidogenic proteins; unlike X-ray crystallography and solution NMR spectroscopy, applications of solid-state NMR spectroscopy are not limited by non-crystalline, non-soluble nature or molecular size of membrane-associated biomolecules. This review article focuses on the applications of solid-state NMR techniques to study a few selected antibacterial and amyloid peptides. Solid-state NMR studies revealing the membrane inserted bent α-helical structure associated with the hemolytic activity of bee venom melittin and the chemical shift oscillation analysis used to determine the transmembrane structure (with α-helix and 310-helix in the N- and C-termini, respectively) of antibiotic peptide alamethicin are discussed in detail. Oligomerization of an amyloidogenic islet amyloid polypeptide (IAPP, or also known as amylin) resulting from its aggregation in a membrane environment, molecular interactions of the antifungal natural product amphotericin B with ergosterol in lipid bilayers, and the mechanism of lipid raft formation by sphingomyelin studied using solid state NMR methods are also discussed in this review article. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato..
9. Takaaki Matsufuji, Masanao Kinoshita, Anna Möuts, J. Peter Slotte, Nobuaki Matsumori, Preparation and Membrane Properties of Oxidized Ceramide Derivatives, Langmuir, 10.1021/acs.langmuir.7b02654, 34, 1, 465-471, 2018.01, Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO-, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes..
10. Yuichi Umegawa, Nobuaki Matsumori, Michio Murata, Recent Solid-State NMR Studies of Hydrated Lipid Membranes, Annual Reports on NMR Spectroscopy, 10.1016/bs.arnmr.2017.12.003, 2018.01, Interactional and structural analyses of lipids in hydrated biomembranes are at the frontier of membrane physics and biology. Recently, solid-state NMR has emerged as a frequently used technique for the investigation of biomembrane systems, leading to particularly remarkable advances in the study of the structural biology of membrane proteins. However, conformational and interactional analyses of lipid molecules and membrane-active small compounds remain challenging. This chapter highlights recent applications of solid-state NMR to membrane lipids and nonpeptidic molecules such as membrane-active natural products. Lipid rafts are microdomains in cellular membranes formed by sphingomyelin and cholesterol and are thought to constitute a platform for signal transduction. Amphotericin B, theonellamide-A, and amphidinol 3 exert their activities by interacting with lipid membranes. Deuterium quadrupole coupling combined with dipole-dipole interactions has been used to evaluate the interaction modes and dynamic properties of membrane lipids and small membrane-active compounds. Herein, we review the recent advances in these topics using our recent research studies as examples of solid-state NMR investigations of hydrated lipid bilayers..
11. Yuma Wakamiya, Makoto Ebine, Mariko Murayama, Hiroyuki Omizu, Nobuaki Matsumori, Michio Murata, Tohru Oishi, Synthesis and Stereochemical Revision of the C31-C67 Fragment of Amphidinol3, Angewandte Chemie - International Edition, 10.1002/anie.201712167, 2018.01, Amphidinol3 (AM3) is a marine natural product produced by the dinoflagellate Amphidinium klebsii. Although the absolute configuration of AM3 was determined in 1999 by extensive NMR analysis and degradation of the natural product, it was a daunting task because of the presence of numerous stereogenic centers on the acyclic carbon chain and the limited availability from natural sources. Thereafter, revisions of the absolute configurations at C2 and C51 were reported in 2008 and 2013, respectively. Reported herein is the revised absolute configuration of AM3: 32S, 33R, 34S, 35S, 36S, and 38S based on the chemical synthesis of partial structures corresponding to the C31-C67 fragment of AM3 in combination with degradation of the natural product. The revised structure is unique in that both antipodal tetrahydropyran counterparts exist on a single carbon chain. The structural revision of AM3 may affect proposed structures of congeners related to the amphidinols..
12. Masayuki Iwamoto, Ayumi Sumino, Eri Shimada, Masanao Kinoshita, Nobuaki Matsumori, Shigetoshi Oiki, Channel Formation and Membrane Deformation via Sterol-Aided Polymorphism of Amphidinol, Scientific Reports, 10.1038/s41598-017-11135-x, 7, 1, 2017.12, Amphidinol 3 (AM3) is an anti-fungal polyene extracted from a marine dinoflagellate. Here, we examined the ion channel activity and membrane-embedded structure of AM3 using a lipid bilayer method and atomic force microscopy (AFM). AM3 exhibited large-conductance (∼1 nS) and non-selective single-channel activity only when sterols were present in the membrane leaflet of the AM3-added side. The variable conductance suggests the formation of a multimeric barrel-stave pore. At high AM3 concentrations, giant-conductance "jumbo" channels (∼40 nS) emerged. AFM revealed a thicker raft-like membrane phase with the appearance of a wrinkled surface, in which phase pores (diameter: ∼10 nm) were observed. The flip-flop of ergosterol occurred only after the appearance of the jumbo channel, indicating that the jumbo channel induced a continuity between the outer and inner leaflets of the membrane: a feature characteristic of toroidal-like pores. Thus, AM3 forms different types of sterol-aided polymorphic channels in a concentration dependent manner..
13. Masanao Kinoshita, Hikaru Ano, Michio Murata, Kenta Shigetomi, Junichi Ikenouchi, Nobuaki Matsumori, Emphatic visualization of sphingomyelin-rich domains by inter-lipid FRET imaging using fluorescent sphingomyelins, Scientific Reports, 10.1038/s41598-017-16361-x, 7, 1, 2017.12, Imaging the distribution of sphingomyelin (SM) in membranes is an important issue in lipid-raft research. Recently we developed novel fluorescent SM analogs that exhibit partition and dynamic behaviors similar to native SM, and succeeded in visualizing lateral domain-segregation between SM-rich liquid-ordered (Lo) and SM-poor liquid-disordered (Ld) domains. However, because the fluorescent contrast between these two domains depends directly on their partition ratio for the fluorescent SMs, domain-separation becomes indeterminate when the distribution difference is not great enough. In this study, we propose the use of inter-lipid Förster resonance energy transfer (FRET) imaging between fluorescent SMs to enhance the contrast of the two domains in cases in which the inter-domain difference in SM distribution is inadequate for conventional monochromic imaging. Our results demonstrate that inter-lipid FRET intensity was significantly higher in the Lo domain than in the Ld domain, resulting in a clear and distinguishable contrast between the two domains even in poorly phase-separated giant unilamellar vesicles. In addition, we show that inter-lipid FRET imaging is useful for selective visualization of highly condensed assemblies and/or clusters of SM molecules in living cell membranes. Thus, the inter-lipid FRET imaging technique can selectively emphasize the SM-condensed domains in both artificial and biological membranes..
14. Ryo Ohtani, Tsukasa Tokita, Tomohisa Takaya, Koichi Iwata, Masanao Kinoshita, Nobuaki Matsumori, Masaaki Nakamura, Leonard F. Lindoy, Shinya Hayami, The impact of metal complex lipids on viscosity and curvature of hybrid liposomes, Chemical Communications, 10.1039/c7cc07944c, 53, 99, 13249-13252, 2017.12, A morphology transformation of hybrid liposomes was shown to occur from spherical vesicles to tubular micelles when increasing the ratio of the metal complex lipid present. Phase transition temperatures increased while viscosities decreased, indicating that the hybrids exhibit stronger interaction between heads but weaker interaction between alkyl chains than occurs in pristine liposomes..
15. Masayuki Satake, Kimberly Cornelio, Shinya Hanashima, Raymond Malabed, Michio Murata, Nobuaki Matsumori, Huiping Zhang, Fumiaki Hayashi, Shoko Mori, Jong Souk Kim, Chang Hoon Kim, Jong Soo Lee, Structures of the Largest Amphidinol Homologues from the Dinoflagellate Amphidinium carterae and Structure-Activity Relationships, Journal of Natural Products, 10.1021/acs.jnatprod.7b00345, 80, 11, 2883-2888, 2017.11, Amphidinols are polyketide metabolites produced by marine dinoflagellates and are chiefly composed of a long linear chain with polyol groups and polyolefins. Two new homologues, amphidinols 20 (AM20, 1) and 21 (AM21, 2), were isolated from Amphidinium carterae collected in Korea. Their structures were elucidated by detailed NMR analyses as amphidinol 6-type compounds with remarkably long polyol chains. Amphidinol 21 (2) has the longest linear structure among the amphidinol homologues reported so far. The congeners, particularly amphidinol 21 (2), showed weaker activity in hemolysis and antifungal assays compared to known amphidinols..
16. Nobuaki Matsumori, Michio Murata, NMR studies on natural product-stereochemical determination and conformational analysis in solution and in membrane, Experimental Approaches of NMR Spectroscopy Methodology and Application to Life Science and Materials Science, 10.1007/978-981-10-5966-7_14, 383-414, 2017.11, In this chapter we overview two topics on NMR methodologies for small molecules, mostly natural products; one is about the solution NMR-based methods used for stereochemical determination of natural products, and the other is on the solid-state and other techniques for investigating natural product-membrane interactions. Since important two methods for stereochemical analysis of natural products, namely the J-based configuration analysis (JBCA) and universal NMR database (UDB) methods, were reported in the 1990s, both methods have been widely used in the field of natural products. The newly coming RDC method is not the major method in the field of natural products yet, but will surely be an important tool for the stereochemical correlation between distant stereogenic centers, which could provide invaluable information as to the whole shape of natural products in solution. In the latter part of this chapter, we discuss the application of solid-state and other NMR techniques to membrane interaction analysis of natural products. In particular, we describe three examples of natural products that interact with biological membranes such as amphotericin B, erythromycin A, and theonellamide A. As shown in NMR studies of amphotericin B, natural products often reveal very high affinities for phospholipids and sterols in bilayer membranes. Solid-state NMR, therefore, provides a very promising approach toward the structure study of membrane-active complexes formed by natural products that have high affinity to lipids. In addition, solution NMR techniques can be applied to elucidate the structural features of membrane-bound small molecules such as antibiotic erythromycin A and membrane-disrupting cyclic peptide theonellamide A. Standard 2D 1H-1H experiments such as COSY (correlation spectroscopy), NOESY (nuclear Overhauser effect spectroscopy), and DOSY (diffusion-ordered spectroscopy) are often helpful in elucidating the membrane interaction between natural products and lipids..
17. Masanao Kinoshita, Kenichi G.N. Suzuki, Nobuaki Matsumori, Misa Takada, Hikaru Ano, Kenichi Morigaki, Mitsuhiro Abe, Asami Makino, Toshihide Kobayashi, Koichiro M. Hirosawa, Takahiro K. Fujiwara, Akihiro Kusumi, Michio Murata, Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs, Journal of Cell Biology, 10.1083/jcb.201607086, 216, 4, 1183-1204, 2017.04, Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone-dependent manners, and that, for ~10-50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size-, cholesterol-, and GPI anchoring-dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM..
18. Takanori Nakane, Shinya Hanashima, Mamoru Suzuki, Haruka Saiki, Taichi Hayashi, Keisuke Kakinouchi, Shigeru Sugiyama, Satoshi Kawatake, Shigeru Matsuoka, Nobuaki Matsumori, Eriko Nango, Jun Kobayashi, Tatsuro Shimamura, Kanako Kimura, Chihiro Mori, Naoki Kunishima, Michihiro Sugahara, Yoko Takakyu, Shigeyuki Inoue, Tetsuya Masuda, Toshiaki Hosaka, Kensuke Tono, Yasumasa Joti, Takashi Kameshima, Takaki Hatsui, Makina Yabashi, Tsuyoshi Inoue, Osamu Nureki, So Iwata, Michio Murata, Eiichi Mizohata, Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1602531113, 113, 46, 13039-13044, 2016.11, The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams..
19. Yasuo Nakagawa, Yuichi Umegawa, Naohiro Matsushita, Tomoya Yamamoto, Hiroshi Tsuchikawa, Shinya Hanashima, Tohru Oishi, Nobuaki Matsumori, Michio Murata, The Structure of the Bimolecular Complex between Amphotericin B and Ergosterol in Membranes Is Stabilized by Face-to-Face van der Waals Interaction with Their Rigid Cyclic Cores, Biochemistry, 10.1021/acs.biochem.6b00193, 55, 24, 3392-3402, 2016.06, Amphotericin B (AmB) is a polyene macrolide antibiotic isolated from Streptomyces nodosus. The antifungal activity of AmB can be attributed to the formation of an ion-channel assembly in the presence of ergosterol (Erg), in which there are two different AmB-Erg orientations, parallel and antiparallel, as reported previously. In this study, to elucidate the structures of those AmB-Erg complexes based on solid-state nuclear magnetic resonance, a 19F-labeled AmB derivative was newly prepared by a hybrid synthesis that utilized degradation products from the drug. Using the 2-(trimethylsilyl)ethoxymethyl (SEM) group as the protecting group for the carboxylic acid moiety of AmB, the fully deprotected labeled AmB compounds were obtained successfully. Then, these labeled AmBs were subjected to 13C{19F} rotational-echo double-resonance (REDOR) experiments in hydrated lipid bilayers. The results indicated the coexistence of parallel and antiparallel orientations for AmB and Erg pairing, at a ratio of 7:3. A total of six distances between AmB and Erg were successfully obtained. Geometry analysis using the distance constraints derived from the REDOR experiments provided the plausible AmB-Erg complex structure for both the parallel and antiparallel interactions. The flat macrolide of AmB and the tetracyclic core of Erg closely contacted in a face-to-face manner, thus maximizing the van der Waals interaction between the two molecules. This interaction can be attributed to the coexistence of both the parallel and antiparallel orientations..
20. Rafael Atillo Espiritu, Kimberly Cornelio, Masanao Kinoshita, Nobuaki Matsumori, Michio Murata, Shinichi Nishimura, Hideaki Kakeya, Minoru Yoshida, Shigeki Matsunaga, Marine sponge cyclic peptide theonellamide A disrupts lipid bilayer integrity without forming distinct membrane pores, Biochimica et Biophysica Acta - Biomembranes, 10.1016/j.bbamem.2016.03.019, 1858, 6, 1373-1379, 2016.06, Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3β-hydroxysterols, resulting in 1,3-β-d-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3β-hydroxyl groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. 31P nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption..
21. Oskar Engberg, Tomokazu Yasuda, Victor Hautala, Nobuaki Matsumori, Thomas K.M. Nyholm, Michio Murata, J. Peter Slotte, Lipid Interactions and Organization in Complex Bilayer Membranes, Biophysical Journal, 10.1016/j.bpj.2015.12.043, 110, 7, 1563-1573, 2016.04, Bilayer lipids influence the lateral structure of the membranes, but the relationship between lipid properties and the lateral structure formed is not always understood. Model membrane studies on bilayers containing cholesterol and various phospholipids (PLs) suggest that high and low temperature melting PLs may segregate, especially in the presence of cholesterol. The effect of different PL headgroups on lateral structure of bilayers is also not clear. Here, we have examined the formation of lateral heterogeneity in increasingly complex (up to five-component) multilamellar bilayers. We have used time-resolved fluorescence spectroscopy with domain-selective fluorescent probes (PL-conjugated trans-parinaric acid), and 2H NMR spectroscopy with site or perdeuterated PLs. We have measured changes in bilayer order using such domain-selective probes both as a function of temperature and composition. Our results from time-resolved fluorescence and 2H NMR showed that in ternary bilayers, acyl chain order and thermostability in sphingomyelin-rich domains were not affected to any greater extent by the headgroup structure of the monounsaturated PLs (phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine) in the bilayer. In the complex five-component bilayers, we could not detect major differences between the different monounsaturated PLs regarding cholesterol-induced ordering. However, cholesterol clearly influenced deuterated N-palmitoyl sphingomyelin differently than the other deuterated PLs, suggesting that cholesterol favored N-palmitoyl sphingomyelin over the other PLs. Taken together, both the fluorescence spectroscopy and 2H NMR data suggest that the complex five-component membranes displayed lateral heterogeneity, at least in the lower temperature regimen examined..
22. Yuichi Umegawa, Yuya Tanaka, Nobuaki Matsumori, Michio Murata, TmDOTA as versatile thermometer compound for solid-state NMR of hydrated lipid bilayer membranes, Magnetic Resonance in Chemistry, 10.1002/mrc.4371, 54, 3, 227-233, 2016.03, Recent advances in solid-state nuclear magnetic resonance (NMR) techniques, such as magic angle spinning and high-power decoupling, have dramatically increased the sensitivity and resolution of NMR. However, these NMR techniques generate extra heat, causing a temperature difference between the sample in the rotor and the variable temperature gas. This extra heating is a particularly crucial problem for hydrated lipid membrane samples. Thus, to develop an NMR thermometer that is suitable for hydrated lipid samples, thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA) was synthesized and labeled with 13C (i.e., 13C-TmDOTA) to increase the NMR sensitivity. The complex was mixed with a hydrated lipid membrane, and the system was subjected to solid-state NMR and differential scanning calorimetric analyses. The physical properties of the lipid bilayer and the quality of the NMR spectra of the membrane were negligibly affected by the presence of 13C-TmDOTA, and the 13C chemical shift of the complex exhibited a large-temperature dependence. The results demonstrated that 13C-TmDOTA could be successfully used as a thermometer to accurately monitor temperature changes induced by 1H decoupling pulses and/or by magic angle spinning and the temperature distribution of the sample inside the rotor. Thus, 13C-TmDOTA was shown to be a versatile thermometer for hydrated lipid assemblies..
23. Kimberly Cornelio, Rafael Atillo Espiritu, Yasuto Todokoro, Shinya Hanashima, Masanao Kinoshita, Nobuaki Matsumori, Michio Murata, Shinichi Nishimura, Hideaki Kakeya, Minoru Yoshida, Shigeki Matsunaga, Sterol-dependent membrane association of the marine sponge-derived bicyclic peptide Theonellamide A as examined by1H NMR, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2016.08.043, 24, 21, 5235-5242, 2016.01, Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several1H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2–9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the1H NMR spectra of TNM-A with sodium dodecyl sulfate-d25(SDS-d25) micelles and small dimyristoylphosphatidylcholine (DMPC)-d54/dihexanoylphosphatidylcholine (DHPC)-d22bicelles in the presence of a paramagnetic quencher Mn2+. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader1H signals of TNM-A were observed in 10 mol % cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn2+to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid–water interface (close to the C2′ portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action..
24. Tomokazu Yasuda, Nobuaki Matsumori, Hiroshi Tsuchikawa, Max Lönnfors, Thomas K M Nyholm, J. Peter Slotte, Michio Murata, Formation of Gel-like Nanodomains in Cholesterol-Containing Sphingomyelin or Phosphatidylcholine Binary Membrane As Examined by Fluorescence Lifetimes and 2H NMR Spectra, Langmuir, 10.1021/acs.langmuir.5b03566, 31, 51, 13783-13792, 2015.12, In this study, we measured the time-resolved fluorescence of trans-parinaric acid (tPA), steady-state fluorescence anisotropy of diphenylhexatriene (DPH), and 2H NMR of 10,10-d2-stearoyl lipids in stearoyl sphingomyelin with cholesterol (SSM/Chol) and l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine with Chol (PSPC/Chol) binary membranes. The results suggest that the membrane order obtained from the fluorescence experiments shows a similar temperature dependency as those of the 2H NMR data. More importantly, the time-resolved fluorescence data implied the presence of at least two types of domains, cholesterol-poor gel-like domains (CPGLD) and cholesterol-enriched liquid-ordered (Lo) domains. These domains appear on a nano-to-micro second time scale for both SSM-Chol and PSPC-Chol membranes. The relative size of the gel-like domain was also estimated from the temperature-dependent lifetime measurements and 2H NMR spectral changes. The results imply that the size of the gel-like domains is very small, probably on the nanometer scale, and smaller in SSM-Chol membrane than those in PSPC-Chol bilayers, which could account for the higher thermal stability of SM-Chol membranes. The present study demonstrates that gel-like nanodomains occur in SM-Chol binary membrane even with Chol content of over 33 mol %, which has been thought to consist exclusively of Lo phase, implying that not only Lo domains but also gel-like nanodomains are important for formation of lipid-ordered phase in SM-Chol and PC-Chol membranes..
25. Michio Murata, Shigeru Sugiyama, Shigeru Matsuoka, Nobuaki Matsumori, Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach, Chemical Record, 10.1002/tcr.201402097, 15, 4, 675-690, 2015.08, Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology. Information on the bioactive structure of lipids is essential to gain deeper insight into biological membranes. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. By examining recent work on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology..
26. 松森 信明, Stereoselective synthesis of the head group of archaeal phospholipid PGP-Me to investigate bacteriorhodopsin-lipid interactions, ORGANIC & BIOMOLECULAR CHEMISTRY, 10.1039/c5ob01252j, 13, 41, 10279-10284, 2015.06.
27. Nobuaki Matsumori, Toshiyuki Yamaguchi, Yoshiko Maeta, Michio Murata, Orientation and Order of the Amide Group of Sphingomyelin in Bilayers Determined by Solid-State NMR, Biophysical Journal, 10.1016/j.bpj.2015.05.011, 108, 12, 2816-2824, 2015.06, Sphingomyelin (SM) and cholesterol (Chol) are considered essential for the formation of lipid rafts; however, the types of molecular interactions involved in this process, such as intermolecular hydrogen bonding, are not well understood. Since, unlike other phospholipids, SM is characterized by the presence of an amide group, it is essential to determine the orientation of the amide and its order in the lipid bilayers to understand the nature of the hydrogen bonds in lipid rafts. For this study, 1′-13C-2-15N-labeled and 2′-13C-2-15N-labeled SMs were prepared, and the rotational-axis direction and order parameters of the SM amide in bilayers were determined based on 13C and 15N chemical-shift anisotropies and intramolecular 13C-15N dipole coupling constants. Results revealed that the amide orientation was minimally affected by Chol, whereas the order was enhanced significantly in its presence. Thus, Chol likely promotes the formation of an intermolecular hydrogen-bond network involving the SM amide without significantly changing its orientation, providing a higher order to the SM amide. To our knowledge, this study offers new insight into the significance of the SM amide orientation with regard to molecular recognition in lipid rafts, and therefore provides a deeper understanding of the mechanism of their formation..
28. Tomokazu Yasuda, Hiroshi Tsuchikawa, Michio Murata, Nobuaki Matsumori, Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution, Biophysical Journal, 10.1016/j.bpj.2015.04.008, 108, 10, 2502-2506, 2015.05, In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (Lo) and liquid-disordered (Ld) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting Lo and Ld domains. We also found that the quadrupole profile of the Lo phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that 2H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the Lo and Ld domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our 2H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation..
29. Tomoya Yamamoto, Yuichi Umegawa, Hiroshi Tsuchikawa, Nobuaki Matsumori, Shinya Hanashima, Michio Murata, Resul Haser, Bernard J. Rawlings, Patrick Caffrey, Role of polyol moiety of amphotericin B in ion channel formation and sterol selectivity in bilayer membrane, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2015.07.009, 23, 17, 5782-5788, 2015.04, Amphotericin B (AmB) is a polyene macrolide antibiotic widely used to treat mycotic infections. In this paper, we focus on the role of the polyol moiety of AmB in sterol selectivity using 7-oxo-AmB, 7α-OH-AmB, and 7β-OH-AmB. The 7-OH analogs were prepared from 7-oxo-AmB. Their K+ flux activity in liposomes showed that introduction of an additional ketone or hydroxy group on the polyol moiety reduces the original activity. Conformational analyses of these derivatives indicated that intramolecular hydrogen-bonding network possibly influenced the conformational rigidity of the macrolactone ring, and stabilized the active conformation in the membrane. Additionally, the flexible polyol leads to destabilization of the whole macrolactone ring conformation, resulting in a loss of sterol selectivity..
30. Yasuo Nakagawa, Yuichi Umegawa, Kenichi Nonomura, Naohiro Matsushita, Tetsuro Takano, Hiroshi Tsuchikawa, Shinya Hanashima, Tohru Oishi, Nobuaki Matsumori, Michio Murata, Axial hydrogen at C7 position and bumpy tetracyclic core markedly reduce sterol's affinity to amphotericin B in membrane, Biochemistry, 10.1021/bi5012942, 54, 2, 303-312, 2015.01, The interaction of amphotericin B (AmB) with fungal ergosterol (Erg) is stronger than its interaction with mammalian cholesterol (Cho), and this property of AmB as an antifungal drug is thought to be responsible for its selective toxicity toward fungi. However, the mechanism by which AmB recognizes the structural differences between sterols, particularly minor difference in the sterol alicyclic portion, is largely unknown. Thus, to investigate the mode of interaction between AmB and the sterol core, we assessed the affinity of AmB to various sterols with different alicyclic structures. Ion flux assays and UV spectral measurements clearly revealed the importance of the Δ7-double bond of the sterol B-ring for interaction with the drug. AmB showed lower affinity for triene sterols, which have double bonds at the Δ5, Δ7, and Δ9 positions. Intermolecular distance measurements by 13C{19F} rotational echo double resonance (REDOR) revealed that the AmB macrolide ring is in closer contact with the steroid core of Erg than it is with the Cho core in the membrane. Conformational analysis suggested that an axial hydrogen atom at C7 of Δ5-sterol (2, 6) and the protruded A-ring of Δ5,7,9-sterol (4, 8) sterically hampered face-to-face contact between the van der Waals surface of the sterol core and the macrolide of AmB. These results further suggest that the α-face of sterol alicycle interacts with the flat macrolide structure of AmB..
31. Jin Cui, Satoshi Kawatake, Yuichi Umegawa, Sébastien Lethu, Masaki Yamagami, Shigeru Matsuoka, Fuminori Sato, Nobuaki Matsumori, Michio Murata, Erratum
Stereoselective synthesis of the head group of archaeal phospholipid PGP-Me to investigate bacteriorhodopsin-lipid interactions (Organic and Biomolecular Chemistry (015) DOI:10.1039/ c5ob01252j), Organic and Biomolecular Chemistry, 10.1039/c5ob90166a, 13, 42, 2015.01.
32. 松森 信明, Direct and Stereospecific Interaction of Amphidinol 3 with Sterol in Lipid Bilayers, BIOCHEMISTRY, 10.1021/bi5002932, 53, 20, 3287-3293, 2014.05.
33. 松森 信明, Coexistence of two liquid crystalline phases in dihydrosphingomyelin and dioleoylphosphatidylcholine binary mixtures, BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 10.1016/j.bbamem.2014.01.017, 1838, 5, 1372-1381, 2014.05.
34. 松森 信明, Effect of Sterol Side Chain on Ion Channel Formation by Amphotericin B in Lipid Bilayers, BIOCHEMISTRY, 10.1021/bi500122c, 53, 19, 3088-3094, 2014.05.
35. 松森 信明, Design and Synthesis of 24-Fluorinated Bafilomycin Analogue as an NMR Probe with Potent Inhibitory Activity to Vacuolar-type ATPase, CHEMISTRY LETTERS, 10.1246/cl.131099, 43, 4, 474-476, 2014.04.
36. 松森 信明, Structure and Biological Activity of 8-Deoxyheronamide C from a Marine-Derived Streptomyces sp.: Heronamides Target Saturated Hydrocarbon Chains in Lipid Membranes, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 10.1021/ja500128u, 136, 14, 5209-5212, 2014.04.
37. 松森 信明, Detailed Comparison of Deuterium Quadrupole Profiles between Sphingomyelin and Phosphatidylcholine Bilayers, BIOPHYSICAL JOURNAL, 10.1016/j.bpj.2013.12.034, 106, 3, 631-638, 2014.02.
38. Nobuaki Matsumori, Structure and interaction in lipid bilayers analyzed using bicelles, Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry, 10.5059/yukigoseikyokaishi.72.596, 72, 5, 596-603, 2014.01, Although many non-peptidic drugs target biological membrane and membrane proteins, it is still difficult to determine the membrane-bound conformation of the drugs. To solve those problems, we have utilized bicelles as a membrane model, since the bicelles, which have planar lipid bilayer portions, are thought to be a more appropriate membrane model than micelles. Small-sized bicelles allow for liquid NMR measurements due to isotropic fast tumbling in solution. We have applied small bicelles to erythromycin A, salinomycin, and amphidinol 3, and determined their membrane-bound structures as well as their positions and orientations in the membranes using coupling constants, NOEs, and paramagnetic relaxation methods. Recently, we found that sphingomyelin, a major lipid constituent of lipid rafts, also forms bicelles, and established its conformation in the bicelles. These studies show the general utility of small bicelles for detailed conformation and orientation analysis of membrane-associated drugs and lipid molecules. We are now developing a bicelle-based crystallization method for membrane proteins, which will facilitate the cocrystalization of membrane proteins and hydrophobic drugs..
39. Makoto Ebine, Mitsunori Kanemoto, Yoshiyuki Manabe, Yosuke Konno, Ken Sakai, Nobuaki Matsumori, Michio Murata, Tohru Oishi, Synthesis and structure revision of the C43-C67 part of amphidinol 3, Organic Letters, 10.1021/ol401176a, 15, 11, 2846-2849, 2013.06, Stereoselective synthesis of the C43-C67 part of amphidinol 3 (AM3) and its C51-epimer was achieved starting from a common intermediate corresponding to the tetrahydropyran moiety of AM3, via asymmetric oxidations and Julia-Kocienski olefination. By comparing NMR data of the synthetic specimens with those of AM3, the absolute configuration at C51 of AM3 was revised from R to S..
40. Masanao Kinoshita, Sarah Goretta, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata, Characterization of the ordered phase formed by sphingomyelin analogues and cholesterol binary mixtures, Biophysics (Japan), 10.2142/biophysics.9.37, 9, 37-49, 2013.05, The influences of structural alterations of sphingomyelin (SM) on its interactions with cholesterol (chol) and on ordered phase formation were examined by density measurements and surface pressure vs. molecular area isotherm measurements. In addition, we quantitatively characterized the ordered phase formed in each SM and chol binary mixture on the basis of the molecular compressional modulus of SM (Cmol-1). Density measurements demonstrated that the ordered phase formation in threo- SM (tSM)/chol and dihydrosphingomyelin (DHSM)/chol binary bilayers shows similar chol concentration-dependency to that of natural erythro-SM (eSM)/chol bilayers; the ordered phase formation was completed in the presence of 25 mol% chol. In contrast, SM bearing a triple bond in the place of a double bond (tripleSM) required a greater concentration of chol to completely transform the bilayer into the ordered phase (at 40mol% chol). Surface pressure vs. molecular area isotherms showed that the DHSM molecule (Cmol-1=290mN/m) is more rigid than eSM (Cmol-1=240mN/m) above 30 mol% chol (in the ordered phase), although these values are similar (140- 150mN/m) in the absence of chol (liquid condensed phase). Most likely, the DHSM/chol mixture forms a more ordered membrane than the eSM/chol mixture does. Moreover, in the absence of chol, the rigidity of the tripleSM molecule (Cmol-1=250mN/m) is significantly higher as compared with that of the eSM molecule (Cmol-1=150mN/m), which is probably due to the presence of a triple bond..
41. Rafael Atillo Espiritu, Nobuaki Matsumori, Michio Murata, Shinichi Nishimura, Hideaki Kakeya, Shigeki Matsunaga, Minoru Yoshida, Interaction between the marine sponge cyclic peptide theonellamide a and sterols in lipid bilayers as viewed by surface plasmon resonance and solid-state 2H nuclear magnetic resonance, Biochemistry, 10.1021/bi4000854, 52, 14, 2410-2418, 2013.04, Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it has been shown that TNMs recognize 3β-hydroxysterol- containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of sterol binding at a molecular level has not been determined. In this study, to gain insight into the mechanism of sterol recognition of TNM in lipid bilayers, surface plasmon resonance (SPR) experiments and solid-state deuterium nuclear magnetic resonance (2H NMR) measurements were performed on theonellamide A (TNM-A). SPR results revealed that the incorporation of 10 mol % cholesterol or ergosterol into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes significantly enhances the affinity of the peptide for the membrane, particularly in the initial binding to the membrane surface. These findings, together with the fact that binding of TNM-A to epicholesterol (3α-cholesterol)-containing liposomes and pure POPC liposomes was comparably weak, confirmed the preference of the peptide for the 3β-hydroxysterol-containing membranes. To further establish the formation of the complex of TNM-A with 3β-hydroxysterols in lipid bilayers, solid-state 2H NMR measurements were conducted using deuterium-labeled cholesterol, ergosterol, or epicholesterol. The 2H NMR spectra showed that TNM-A significantly inhibits the fast rotational motion of cholesterol and ergosterol, but not epicholesterol, therefore verifying the direct complexation between TNM-A and 3β-hydroxysterols in lipid bilayers. This study demonstrates that TNM-A directly recognizes the 3β-OH moiety of sterols, which greatly facilitates its binding to bilayer membranes..
42. Nobuaki Matsumori, Yuki Hiradate, Hajime Shibata, Tohru Oishi, Shuichi Shimma, Michisato Toyoda, Fumiaki Hayashi, Manabu Yoshida, Michio Murata, Masaaki Morisawa, A novel sperm-activating and attracting factor from the ascidian Ascidia sydneiensis, Organic Letters, 10.1021/ol303172n, 15, 2, 294-297, 2013.01, A novel SAAF was isolated from the title ascidian. The structure was elucidated using the entire sample of 4 nmol, suggesting that the position of the OH group confers genus-specificity to sperm chemotaxis in ascidians. This study not only provides insight into the chemical tactics in sperm chemotaxis but demonstrates that the innovative techniques allow structure determination of natural products in trace amounts..
43. Yoshiyuki Manabe, Makoto Ebine, Nobuaki Matsumori, Michio Murata, Tohru Oishi, Confirmation of the absolute configuration at C45 of amphidinol 3, Journal of Natural Products, 10.1021/np300604w, 75, 11, 2003-2006, 2012.12, Amphidinol 3 (AM3), a membrane-active agent isolated from the dinoflagellate Amphidinium klebsii, consists of a long carbon chain containing 25 stereogenic centers. Although the absolute configuration of AM3 was determined by extensive NMR analysis and degradation of the natural product, the partial structure corresponding to the tetrahydropyran ring system was found to be antipodal to that of karlotoxin 2, a structurally related compound recently isolated from the dinoflagellate Karlodinium veneficum. By extensive degradation of the natural product and conversion of the resulting alcohol to an MTPA ester, the absolute configuration at C45 of AM3 was confirmed to be R, supporting the originally proposed structure..
44. Yuichi Umegawa, Takeshi Adachi, Nobuaki Matsumori, Michio Murata, Possible conformation of amphotericin B dimer in membrane-bound assembly as deduced from solid-state NMR, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2012.08.016, 20, 19, 5699-5704, 2012.10, Aiming for structural analysis of amphotericin B (AmB) ion-channel assemblies in membrane, a covalent dimer was synthesized between 13C-labled AmB methyl ester and 19F-labled AmB. The dimer showed slightly weaker but significant biological activities against fungi and red blood cells compared with those of monomeric AmB. Then the dimer was subjected to 13C{19F}REDOR (Rotational-Echo Double Resonance) experiments in hydrated lipid bilayers. The obtained REDOR dephasing effects were explained by two components; a short 13C/19F distance (6.9 Å) accounting for 23% of the REDOR dephasing, and a longer one (14 Å) comprising the rest of the dephasing. The shorter distance is likely to reflect the formation of barrel-stave ion channel..
45. Sarah A. Goretta, Masanao Kinoshita, Shoko Mori, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata, Effects of chemical modification of sphingomyelin ammonium group on formation of liquid-ordered phase, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2012.05.015, 20, 13, 4012-4019, 2012.07, Sphingomyelin (SM) and cholesterol form microdomains called lipid rafts in cellular membranes. To develop a versatile fluorescent lipid probe, chemical modifications to both the hydrophobic and hydrophilic portions of SM are essential. Few reports describing SM probes with a fluorophore at the polar head group have been published. This study examined the effect of substitution on an ammonium moiety of SM on the membrane properties of SM. Two SM analogs with small propargyl and allyl groups on the quaternary nitrogen atom were synthesized and subjected to analysis using differential scanning calorimetry, fluorescent anisotropy, detergent solubilization, surface pressure, and density measurements. Results demonstrated that the two SM analogs retained the membrane properties of SM, including formation of an ordered phase and the ability to interact with cholesterol. A dansyl-substituted SM was prepared for fluorescent measurements. Dansyl-SM showed less of a propensity to form microdomains. These findings imply the potential application of N-substituted SMs as a raft-specific molecular probe..
46. Tohru Oishi, Keiichi Konoki, Rie Tamate, Kohei Torikai, Futoshi Hasegawa, Nobuaki Matsumori, Michio Murata, Artificial ladder-shaped polyethers that inhibit maitotoxin-induced Ca 2+ influx in rat glioma C6 cells, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2012.04.053, 22, 11, 3619-3622, 2012.06, Maitotoxin (MTX) is a ladder-shaped polyether produced by the epiphytic dinoflagellate Gambierdiscus toxicus. It is known to elicit potent toxicity against mammals and induce influx of Ca2+ into cells. An artificial ladder-shaped polyether possessing a 6/7/6/6/7/6/6 heptacyclic ring system, which was designed for elucidating interactions with transmembrane proteins, was found to be the most potent inhibitor against MTX-induced Ca2+ influx that has ever been reported..
47. Yuichi Umegawa, Yasuo Nakagawa, Kazuaki Tahara, Hiroshi Tsuchikawa, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Head-to-tail interaction between amphotericin b and ergosterol occurs in hydrated phospholipid membrane, Biochemistry, 10.1021/bi2012542, 51, 1, 83-89, 2012.01, Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed 13C{ 19F}REDOR experiments with C14-19F-labeled AmB and biosynthetically 13C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, 13C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, 13C{ 31P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface..
48. Toshiyuki Yamaguchi, Takashi Suzuki, Tomokazu Yasuda, Tohru Oishi, Nobuaki Matsumori, Michio Murata, NMR-based conformational analysis of sphingomyelin in bicelles, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2011.11.001, 20, 1, 270-278, 2012.01, Sphingomyelin (SM) is a common sphingolipid in mammalian membranes and is known to be substantially involved in cellular events such as the formation of lipid rafts. Despite its biological significance, conformation of SM in a membrane environment remains unclear because the noncrystalline property and anisotropic environment of lipid bilayers hampers the application of X-ray crystallography and NMR measurements. In this study, to elucidate the conformation of SM in membranes, we utilized bicelles as a substitute for a lipid bilayer membrane. First, we demonstrated through 31P NMR, 2H NMR, and dynamic light scattering experiments that SM forms both oriented and isotropic bicelles by changing the ratio of SM/dihexanoyl phosphatidylcholine. Then, we determined the conformation of SM in isotropic bicelles on the basis of coupling constants and NOE correlations in 1H NMR and found that the C2-C6 and amide groups of SM take a relatively rigid conformation in bicelles..
49. Nobuaki Matsumori, Norio Tanada, Kohei Nozu, Hiroki Okazaki, Tohru Oishi, Michio Murata, Design and synthesis of sphingomyelin-cholesterol conjugates and their formation of ordered membranes, Chemistry - A European Journal, 10.1002/chem.201100849, 17, 31, 8568-8575, 2011.07, A lipid raft is a cholesterol (Chol)-rich microdomain floating in a sea of lipid bilayers. Although Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM), rather than with glycerophospholipids, the origin of the specific interaction has remained unresolved, primarily because of the high mobility of lipid molecules and weak intermolecular interactions. In this study, we synthesized SM-Chol conjugates with functionally designed linker portions to restrain Chol mobility and examined their formation of ordered membranes by a detergent insolubility assay, fluorescence anisotropy experiments, and fluorescence-quenching assay. In all of the tests, membranes prepared from the conjugates showed properties of ordered domains comparable to a SM-Chol (1:1) membrane. To gain insight into the structure of bilayers composed from the conjugates, we performed molecular dynamics simulations with 64 molecules of the conjugates, which suggested that the conjugates form a stable bilayer structure by bending at the linker portion and, mostly, reproduce the hydrogen bonds between the SM and Chol portions. These results imply that the molecular recognition between SM and Chol in an ordered domain is essentially reproduced by the conjugated molecules and, thus, demonstrates that these conjugate molecules could potentially serve as molecular probes for understanding molecular recognition in lipid rafts..
50. Minako Hirano, Yuko Takeuchi, Nobuaki Matsumori, Michio Murata, Toru Ide, Channels formed by amphotericin B covalent dimers exhibit rectification, Journal of Membrane Biology, 10.1007/s00232-011-9354-x, 240, 3, 159-164, 2011.04, Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification..
51. Keisuke Maruyoshi, Toshiyuki Yamaguchi, Tetsuo Demura, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Conformations of spermine in adenosine triphosphate complex
The structural basis for weak bimolecular interactions of major cellular electrolytes, Chemistry - A European Journal, 10.1002/chem.201002759, 17, 17, 4788-4795, 2011.04, Selectively 2H- and 13C-labeled spermines (SPM) were efficiently synthesized and analyzed by NMR spectroscopy to determine the spin-spin coupling constants for six conformationally relevant bonds. SPM that is composed of three alkyl moieties, a butanylene, and two propanylene chains undergoes a conformational change when interacting with multivalent anions (e.g., adenosine triphosphate (ATP), ATP-Mg2+, and tripolyphosphate). Upon interaction with ATP, the C-C bonds, which affect the distance between the neighboring pairs of ammonium groups (i.e., N1/N5 and N5/N5′), increase the population of gauche rotamers by 17-20 % relative to those in the 4 HCl salt of SPM. However, the trend in increments of the gauche conformers for the SPM-ATP complex profoundly differs from that of the spermidine (SPD)-ATP complex. This implies that SPM may preferentially recognize the adenyl group of ATP rather than the tripolyphosphate moiety. This may account for the higher affinity of SPM to ATP-Mg2+ than with that of SPD, which chiefly interacts with β- and Î-phosphates and is easily replaced by Mg 2+. These results may provide a clue for the further understanding of the structural basis of polyamine biological functions. Spermines and spin: Spermines (SPM) labeled selectively with 2H and 13C were used to determine the spin-spin coupling constants for six conformationally relevant bonds. SPM revealed diverse conformational changes upon interaction with adenosine triphosphate (ATP), ATP-Mg2+, and tripolyphosphate (TPP, see graph)..
52. Asuka Kitamura, Kouta Matsui, Keiichi Konoki, Nobuaki Matsumori, Michio Murata, Toru Kawakami, Saburo Aimoto, Lysine proximity significantly affects glycation of lysine-containing collagen model peptides, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2011.02.048, 19, 7, 2125-2129, 2011.04, Advanced glycation end products (AGE) are known to cause diabetes complications in hyperglycemia patients. In this study we prepared hetero-trimers of collagen model peptides comprising Ac-(Pro-Hyp-Gly) 5-Pro-Lys-Gly-(Pro-Hyp-Gly)5-Ala-NH2 (4) and Ac-(Pro-Hyp-Gly)11-Ala-NH2 (5) to investigate the clustering effect of lysine on AGE formation. The formation rate of carboxymethyllysine over several months was determined for the mixtures of peptides 4 and 5 at (3:0), (2:1) and (1:2) in the presence of glucose. The contents of carboxymethyllysine were significantly enhanced for (3:0) and (2:1) as compared with (1:2), suggesting that the proximity of lysine residues in the trimers accelerated formation of the AGE. Furthermore, a lysine dimerization moiety (GOLD) was identified for the first time from AGEs of glucose origin, which implied the significance of GOLD in oligomerization of collagens and other long-life proteins..
53. Yusuke Kasai, Nobuaki Matsumori, Hiroyuki Ueno, Kenichi Nonomura, Shinya Yano, Murata Michio, Tohru Oishi, Synthesis of 6-F-ergosterol and its influence on membrane-permeabilization of amphotericin B and amphidinol 3, Organic and Biomolecular Chemistry, 10.1039/c0ob00685h, 9, 5, 1437-1442, 2011.03, Two well-known antifungals, amphotericin B (AmB) and amphodinol 3 (AM3), are thought to exert antifungal activity by forming ion-permeable channels or pores together with sterol molecules. However, detailed molecular recognitions for AmB-sterol and AM3-sterol in lipid bilayers have yet to be determined. Toward 19F NMR-based investigation of the molecular recognition underlying their potent antifungal activity, we synthesized 6-fluoro-ergosterol in five steps via ring opening of (5α,6α)-epoxide of ergosterol acetate with using novel combination of TiF4 and n-Bu 4N+Ph3SiF2-. Then we evaluated its activity of promoting pore formation of AmB and AM3, and found that pore formation of AmB was barely promoted by 6-F-ergosterol in clear contrast to the dramatic promotion effect of unmodified ergosterol, whereas AM3 activity was markedly enhanced in the presence of 6-F-ergosterol, which was comparable to that of unmodified ergosterol. These results indicate that the introduction of an F atom at C6 position of ergosterol plays an inhibitory role in interacting with AmB, but it is not the case with AM3..
54. Nobuaki Matsumori, Horoki Okazaki, Kaoru Nomura, Michio Murata, Fluorinated cholesterol retains domain-forming activity in sphingomyelin bilayers, Chemistry and Physics of Lipids, 10.1016/j.chemphyslip.2011.05.007, 164, 5, 401-408, 2011.01, Lipid rafts are cholesterol (Chol)-rich microdomains floating in a sea of lipid bilayers. Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM) rather than with glycerophospholipids, and this putative SM-Chol interaction is generally recognized as a requirement for raft formation. However, the presence of the specific interaction is still controversial, primarily because of the lack of useful molecular probes for scrutinizing this interaction. Recently, we reported that the dynamic properties of 6-F-Chol in DMPC bilayers are similar to those of unmodified Chol. Hence, in the present study, we first compared the roles of 6-F-Chol and Chol in SM bilayers through detergent insolubility, fluorescence polarization, and 2H NMR experiments. The results demonstrated that 6-F-Chol and Chol behave similarly in SM bilayers, whereas, in SM-DOPC membranes, 6-F-Chol is less effective in domain formation. Then, we analyzed the molecular orientation of 6-F-Chol in SM bilayers using solid-state NMR, and found that the dynamics and orientation of 6-F-Chol in SM bilayers are almost identical to those in DMPC bilayers. This supports the notion of the lack of a putative specific interaction between SM and Chol. Thus, this study demonstrates the utility of 6-F-Chol as a molecular probe for understanding molecular recognition in lipid rafts..
55. Satoru Ujihara, Tohru Oishi, Ryota Mouri, Rie Tamate, Keiichi Konoki, Nobuaki Matsumori, Michio Murata, Yasukatsu Oshima, Naoyuki Sugiyama, Masaru Tomita, Yasushi Ishihama, Detection of Rap1A as a yessotoxin binding protein from blood cell membranes, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2010.09.080, 20, 22, 6443-6446, 2010.11, As is the case with other ladder-shaped polyether compounds, yessotoxin is produced by marine dinoflagellate, and possesses various biological activities beside potent toxicity. To gain a better understanding of the molecular mechanism for high affinity between these polyethers and their binding proteins, which accounts for their powerful biological activities, we searched for its binding proteins from human blood cells by using the biotin-conjugate of desulfated YTX as a ligand. By a protein pull-down protocol with use of streptavidin beads, a band of specifically binding proteins was detected in SDS-PAGE. HPLC-tandem mass spectrometry (MS/MS) indicated that Rap 1A, one of Ras superfamily proteins, binds to the YTX-linked resins. Western blotting and surface plasmon resonance experiments further confirmed that Rap1A specifically binds to YTX with the KD value around 4 μM..
56. Nobuaki Matsumori, Michio Murata, 3D structures of membrane-associated small molecules as determined in isotropic bicelles, Natural Product Reports, 10.1039/c0np00002g, 27, 10, 1480-1492, 2010.10, About half of known bioactive organic molecules, including drugs, are known to target biological membranes and membrane proteins. Despite this, it has proven difficult to define the membrane-bound conformations of these molecules. In recent years, bicelles have been recognized as a more appropriate membrane model than micelles because their planar portion is composed of a lipid bilayer. Bicelles with a small diameter, termed isotropic or fast-tumbling bicelles, allow for high-resolution NMR measurements due to their high mobility in suspension, and therefore have become a versatile tool for structure studies of membrane-associated molecules. Following a brief description of the morphology and preparation of isotropic bicelles, we summarize their application to structural studies of membrane-bound peptides and small molecules, and then highlight our recent studies on the 3D structures of erythromycin A, salinomycin and amphidinol 3 using isotropic bicelles..
57. Respati T. Swasono, Ryota Mouri, Nagy Morsy, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Sterol effect on interaction between amphidinol 3 and liposomal membrane as evidenced by surface plasmon resonance, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2010.02.025, 20, 7, 2215-2218, 2010.04, The affinity of amphidinol 3 (AM3) to phospholipid membranes in the presence and absence of sterol was examined by surface plasmon resonance (SPR) experiments. The results showed that AM3 has 1000 and 5300 times higher affinity for cholesterol- and ergosterol-containing liposomes, respectively, than those without sterol. The two-state reaction model well reproduced the sensor grams, which indicated that the interaction is composed of two steps, which correspond to binding to the membrane and internalization to form stable complexes..
58. Respati T. Swasono, Mitsunori Kanemoto, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Structural reevaluations of amphidinol 3, a potent antifungal compound from dinoflagellate, Heterocycles, 10.3987/COM-10-S(E)86, 82, 2, 1359-1369, 2010.02, Among other homologues, amphidinol 3 (AM3) is the most potent antifungal compound isolated from the dinoflagellate Amphidinium klebsii. AM3 undergoes conformational changes in organic solvents while it takes relatively fixed configuration in a membrane model. By using NMR data of peracetyl AM3, we were able to confirm the configuration of C50-C51 of AM3 which remained uncertain in our previous study..
59. Tetsuro Takano, Keiichi Konoki, Nobuaki Matsumori, Michio Murata, Amphotericin B-induced ion flux is markedly attenuated in phosphatidylglycerol membrane as evidenced by a newly devised fluorometric method, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2009.07.036, 17, 17, 6301-6304, 2009.09, The effect of phospholipid head group on the membrane-permeabilizing activity of amphotericin B (AmB) was examined using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) liposomes. The activity of AmB was evaluated as K+ influx measured as pH change inside liposomes by fluorescent measurements of 2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein (BCECF). AmB showed prominent permeability in POPC liposomes, whereas hardly inducing ion flux in POPG membrane. POPC added to POPG liposomes as a minor constituent markedly enhanced membrane permeability, indicating the importance of a phosphonocholine group of PC for the drug's activity..
60. Nobuaki Matsumori, Kazuaki Tahara, Hiroko Yamamoto, Atsushi Morooka, Mototsugu Doi, Tohru Oishi, Michio Murata, Direct interaction between amphotericin B and ergosterol in lipid bilayers as revealed by 2H NMR spectroscopy, Journal of the American Chemical Society, 10.1021/ja9033473, 131, 33, 11855-11860, 2009.08, Although amphotericin B (AmB) is thought to exert its antifungal activity by forming transmembrane ion-permeable self-assemblies together with ergosterol, no previous study has directly proven AmB-ergosterol interaction. To establish the interaction, we measured 2H NMR using deuterium-labeled sterols and AmB. The 2H NMR spectra of deuterated ergosterol in palmitoyloleoylphosphatidylcholine (POPC) bilayers showed that fast axial diffusion of erogosterol was almost completely inhibited by the coexistence of AmB. Conversely, cholesterol mobility in POPC membrane was essentially unchanged with or without AmB. These results unequivocally demonstrate that ergosterol has significant interaction with AmB in POPC bilayers. In addition, we examined the mobility of AmB using deuterium-labeled AmB, and found that, although AmB is almost immobilized in sterol-free and cholesterol-containing POPC membranes, a certain ratio of AmB molecules acquires mobility in the presence of ergosterol. The similar mobility of AmB and ergosterol in POPC bilayers confirmed the idea of the direct intermolecular interaction between ergosterol and AmB..
61. Michio Murata, Yusuke Kasai, Yuichi Umegawa, Naohiro Matsushita, Hiroshi Tsuchikawa, Nobuaki Matsumori, Tohru Oishi, Ion channel complex of antibiotics as viewed by NMR, Pure and Applied Chemistry, 10.1351/PAC-CON-08-08-37, 81, 6, 1123-1129, 2009.07, Amphotericin B (AmB) exerts its pharmacological effects by forming a barrel-stave assembly in fungal membranes. To examine the interaction between AmB and ergosterol or cholesterol, 13C- and 19F-labeled covalent conjugates were prepared and subjected to solidstate NMR measurements. Using rotor-synchronous double-resonance experiments such as rotational echo double resonance (REDOR) and RDX, we estimated the distance between the fluorine atom and its nearest carbon in the heptaene moiety to be less than 8.6 A ̊, indicating that the B ring of ergosterol comes close to the AmB polyene moiety. Conformational search of the AmB-ergosterol conjugate using the NMR-derived constraints suggested that ergosterol molecules surround the AmB assembly in contrast to the conventional image where ergosterol is inserted into AmB molecules. AmB-AmB bimolecular interaction was examined by using 13C- and 19F-labeled AmBs in dimyritoylphosphatidylcholine (DMPC) membrane without sterols. 13C- and 19F dipolar interactions deriving from both head-to-head and head-to-tail orientations were observed in the REDOR experiments. The interactions between AmB and acyl chains of the phospholipid were also detected..
62. Ryota Mouri, Tohru Oishi, Kohei Torikai, Satoru Ujihara, Nobuaki Matsumori, Michio Murata, Yasukatsu Oshima, Surface plasmon resonance-based detection of ladder-shaped polyethers by inhibition detection method, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2009.03.103, 19, 10, 2824-2828, 2009.05, Ladder-shaped polyether (LSP) compounds represented by brevetoxins and ciguatoxins were largely discovered in association with seafood poisoning. Thus, a quick quantification method for LSPs is potentially important. We examined a surface plasmon resonance method using desulfated-yessotoxin (dsYTX) immobilized on a sensor chip and phosphodiesterase PDEII in a inhibition detection mode. Yessotoxin, brevetoxin B and synthetic LSP derivatives showed clear inhibition against PDEII binding to the immobilized dsYTX, by which their half inhibitory concentrations were successfully estimated. This inhibition method appeared to be superior in specificity to direct binding assays where binding proteins to LSP was immobilized on a sensor chip..
63. Keisuke Maruyoshi, Kaori Nonaka, Takeshi Sagane, Tetsuo Demura, Toshiyuki Yamaguchi, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Conformational change of spermidine upon interaction with adenosine triphosphate in aqueous solution, Chemistry - A European Journal, 10.1002/chem.200801961, 15, 7, 1618-1626, 2009.02, Endogenous polyamines, represented by putrescine, spermidine, and spermine, are known to exert their physiological functions by interacting with polyanionic biomolecules such as DNA, RNA, adenosine triphosphate (ATP), and phospholipids. Very few examples of conformation analysis have been reported for these highly flexible polymethylene compounds, mainly due to the lack of appropriate methodologies. To understand the molecular basis of the weak interaction between polyamines and polyanions that underlies their physiological functions, we aimed to elucidate the solution conformation of spermidine by using diastereospecifically deuterated and
13
C-labeled derivatives (1-7), which were designed to diagnose the orientation of seven conformationally relevant bonds in spermidine. 1H-1H and
13
C-1H NMR coupling constants (
3
J
H,H
and
3
J
C,H
) were successfully determined for a spermidine-ATP complex. The relevant coupling constants markedly decreased upon complexation. The results reveal that spermidine, when interacting with ATP, undergoes changes that make the conformation more bent and forms the complex with the triphosphate part of ATP in an orientation-sensitive manner..
64. Naohiro Matsushita, Yukiko Matsuo, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata, Tohru Oishi, Synthesis of 25- 13c-amphotericin B methyl ester
A molecular probe for solid-state NMR measurements, Chemistry Letters, 10.1246/cl.2009.114, 38, 2, 114-115, 2009, A 13C-labeled amphotericin B (AmB) derivative was synthesized based on a hybrid strategy combining chemical synthesis with degradation of a natural product through successive cross-coupling reactions and macrolactonization. The specimen regiospecifically 13C-labeled (99% enrichment) at C25 position corresponding to the polyene moiety would be a powerful tool for structural analysis of the molecular assembly formed by AmB based on solid-state NMR measurements..
65. Toshiyuki Yamaguchi, Keisuke Marayoshi, Nobuaki Matsumori, Michio Murata, Accurate measurement of vicinal carbon-hydrogen coupling constants via ammonium nitrogen based on HMBC experiments, Chemistry Letters, 10.1246/cl.2008.1172, 37, 11, 1172-1173, 2008.12, Vicinal spin-spin coupling constants frequently used for conformation analysis were determined for ammonium-containing 13C-N-C- 1H systems by using HMBC. 1-Deoxynojirimycin hydrochloride provided an appropriate system for measuring the antiperiplanar and gauche interactions, which were determined to be 7.3 and 1.6 Hz, respectively..
66. Tohru Oishi, Mitsunori Kanemoto, Respati Swasono, Nobuaki Matsumori, Michio Murata, Combinatorial synthesis of the 1,5-polyol system based on cross metathesis
Structure revision of amphidinol 3, Organic Letters, 10.1021/ol802168r, 10, 22, 5203-5206, 2008.12, (Chemical Equation Presented) Combinatorial synthesis of a 1,5-polyol system corresponding to the C1-C14 unit of amphidinol 3 (AM3) and its diastereomers was achieved via chemoselective cross metathesis as the key step. Comparison of 13C NMR data of the synthetic specimens with that of AM3 led to a controversy regarding the originally proposed structure. From GC-MS analysis of the degradation product, the absolute configuration at C2 of AM3 has been revised to be R..
67. Tohru Oishi, Futoshi Hasegawa, Kohei Torikai, Keiichi Konoki, Nobuaki Matsumori, Michio Murata, Convergent synthesis and biological activity of the WXYZA′B′ C′ ring system of maitotoxin, Organic Letters, 10.1021/ol801369g, 10, 16, 3599-3602, 2008.12, (Chemical Equation Presented) The WXYZA′B′C′ ring system (1) of maitotoxin (MTX) was synthesized in a convergent manner via successive coupling of the W, Z, and C′ ring fragments through construction of the XY and A′B′ ring systems. The synthetic segment 1 blocked the hemolytic activity elicited by MTX..
68. Yuichi Umegawa, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Ergosterol increases the intermolecular distance of amphotericin B in the membrane-bound assembly as evidenced by solid-state NMR, Biochemistry, 10.1021/bi801875y, 47, 51, 13463-13469, 2008.12, Amphotericin B (AmB) exerts its antifungal activity by forming ion-permeable assemblies across lipid bilayers. To investigate AmB-AmB bimolecular interactions in the assembly, we carried out 13C{ 19F}REDOR experiments using 14-19F- and 13C41-labeled AmBs in sterol-containing and sterol-free palmitoyloleoylphosphatidylcholine (POPC) membranes and measured the average distance between the labeled sites of AmBs in membrane-bound forms. The REDOR results suggested that the intermolecular distance of AmB molecules is significantly increased in the ergosterol membrane as compared with the cholesterol membrane. This sterol-dependent change was supported by the UV spectra of AmB in lipid bilayers, in which the excitonic absorption band arising from the aggregated state of AmB shifted to longer wavelength in ergosterol-containing POPC membrane. The REDOR experiments also disclosed that the head-to-head orientation of AmB is predominant in both of the sterol-containing membranes and AmB-POPC interaction was detected only in the ergosterol membrane. Ergosterol significantly influences the interactions between AmB molecules as well as those between AmB and POPC, which may facilitate formation of ion-permeable channels in ergosterol-containing membrane..
69. Satoru Ujihara, Tohru Oishi, Kohei Torikai, Keiichi Konoki, Nobuaki Matsumori, Michio Murata, Yasukatsu Oshima, Saburo Aimoto, Interaction of ladder-shaped polyethers with transmembrane α-helix of glycophorin A as evidenced by saturation transfer difference NMR and surface plasmon resonance, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2008.10.020, 18, 23, 6115-6118, 2008.12, Ladder-shaped polyether (LSP) compounds are thought to interact with transmembrane α-helices, but direct evidence has scarcely obtained for these interactions. We adopted a transmembrane α-helix of glycophorin A, and quantitatively evaluated its interaction with LSPs such as yessotoxin (YTX), desulfated YTX and artificial LSPs, using surface plasmon resonance and saturation transfer difference NMR. As a result, dissociation constants (KD) of YTX and desulfated YTX to a transmembrane domain peptide of glycophorin A were determined to be in the submillimolar range. Furthermore, in saturation transfer difference NMR, the signals at the polyene side chain and the angular methyl groups of YTX were significantly attenuated, which probably comprised an interacting interface of LSPs with a transmembrane α-helix. These results suggest that hydrophobic interaction plays an important role in molecular recognition of the α-helix peptide by LSPs..
70. Michio Murata, Nobuaki Matsumori, Keiichi Konoki, Tohru Oishi, Structural features of dinoflagellate toxins underlying biological activity as viewed by NMR, Bulletin of the Chemical Society of Japan, 10.1246/bcsj.81.307, 81, 3, 307-319, 2008.12, Marine dinoflagellates are a rich source of structurally and biologically intriguing secondary metabolites. Among those, maitotoxin may be one of the best known examples, which is the largest natural product known to date and possesses the most potent toxicity known amongst non-proteinaceous compounds. Structural studies of maitotoxin are reviewed with a particular focus on the configuration and mode of action of this unique toxin. In addition, there are many marine natural products which bind to biomembranes to exert their biological activities. To gain a better understanding of their mode of action, conformation, and bimoleeular interaction occurring in biomembranes are particularly important. Recent results from NMR studies of membrane systems in the authors' group are reviewed briefly..
71. Toshihiro Houdai, Nobuaki Matsumori, Michio Murata, Structure of membrane-bound amphidinol 3 in isotropic small bicelles, Organic Letters, 10.1021/ol8016337, 10, 19, 4191-4194, 2008.12, (Chemical Equation Presented) Amphidinol 3 (AM3) exhibits a potent membrane permeabilizing activity by forming pores in biological membranes. We examined the conformation and location of AM3 using Isotropic bicelles, a more natural membrane model than micelles. The results show that AM3 takes turn structures at the two tetrahydropyran rings. Most of the hydrophilic region of the molecule is predominantly present in the surface, while the hydrophobic polyolefin penetrates in the bicelle interior..
72. Kohei Torikai, Tohru Oishi, Satoru Ujihara, Nobuaki Matsumori, Keiichi Konoki, Michio Murata, Saburo Aimoto, Design and synthesis of ladder-shaped tetracyclic, heptacyclic, and decacyclic ethers and evaluation of the interaction with transmembrane proteins, Journal of the American Chemical Society, 10.1021/ja801576v, 130, 31, 10217-10226, 2008.08, Ladder-shaped polyether (LSP) toxins represented by brevetoxins and Ciguatoxins are thought to bind to transmembrane (TM) proteins. To elucidate the interactions of LSPs with TM proteins, we have synthesized artificial ladder-shaped polyethers (ALPs) containing 6/7/6/6 tetracyclic, 6/7/6/6/7/6/6 heptacyclic, and 6/7/6/6/7/6/6/7/6/6 decacyclic systems, based on the convergent method via α-cyano ethers. The ALPs possessing the simple iterative structure with different numbers of rings would be useful for structure-activity relationship studies on the molecular length, which is supposed to be important when naturally occurring LSPs elicit their toxicity. Two series of ALPs were prepared to evaluate the hydrophilic or hydrophobic effects of the side chains: (i) both sides were functionalized as diols (A series), and (ii) one side remained as diol and the other side was protected as benzyl ethers (B series). To examine the interaction of these ALPs with TM proteins, dissociation of glycophorin A (GpA) dimers into monomers was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The heptacyclic ether (ALP7B) elicited the most potent activity in the presence of 2% SDS buffer, whereas the decacyclic ether (ALP10A) exhibited an intriguing phenomenon to induce precipitation of GpA in a dose-dependent manner, under the low concentration of SDS (0.03%). ALP10A also induced precipitation of integrin α 1β1, a TM protein known to form heterodimers in the lipid bilayer membranes. The different activities among the ALPs can be accounted for by the concept of "hydrophobic matching" that is, lengths of the hydrophobic region including the side chains of ALP7B and ALP10A are ca. 25 Å, which match the lengths of the hydrophobic region of α-helical TM proteins, as well as the hydrophobic thickness of lipid bilayer membranes. The concept of the hydrophobic matching would be a clue to understanding the interaction between LSPs and TM proteins, and also a guiding principle to design ALPs possessing potent affinities with TM proteins..
73. Ryota Mouri, Keiichi Konoki, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance, Biochemistry, 10.1021/bi800334p, 47, 30, 7807-7815, 2008.07, Amphotericin B (AmB) is a membrane-active antibiotic that increases the permeability of fungal membranes. Thus, the dynamic process of its interaction with membranes poses intriguing questions, which prompted us to elaborate a quick and reliable method for real-time observation of the drug's binding to phospholipid liposomes. We focused on surface plasmon resonance (SPR) and devised a new modification method of sensor chips, which led to a significant reduction in the level of nonspecific binding of the drug in a control lane. With this method in hand, we examined the affinity of AmB for various membrane preparations. As expected, AmB exhibited much higher affinity for sterol-containing palmitoyloleoylphosphatidylcholine membranes than those without sterol. The sensorgrams recorded under various conditions partly fitted theoretical curves, which were based on three interaction models. Among those, a two-state reaction model reproduced well the sensorgram of AmB binding to an ergosterol-containing membrane; in this model, two states of membrane-bound complexes, AB and AB*, are assumed, which correspond to a simple binding to the surface of the membrane (AB) and formation of another assembly in the membrane (AB*) such as an ion channel complex. Kinetic analysis demonstrated that the association constant in ergosterol-containing POPC liposomes is larger by 1 order of magnitude than that in the cholesterol-containing counterpart. These findings support the previous notion that ergosterol stabilizes the membrane-bound assembly of AmB..
74. Nagy Morsy, Keiichi Konoki, Toshihiro Houdai, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Saburo Aimoto, Roles of integral protein in membrane permeabilization by amphidinols, Biochimica et Biophysica Acta - Biomembranes, 10.1016/j.bbamem.2008.01.018, 1778, 6, 1453-1459, 2008.06, Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers..
75. Nobuaki Matsumori, Yusuke Kasai, Tohru Oishi, Michio Murata, Kaoru Nomura, Orientation of fluorinated cholesterol in lipid bilayers analyzed by 19F tensor calculation and solid-state NMR, Journal of the American Chemical Society, 10.1021/ja077580l, 130, 14, 4757-4766, 2008.04, 6-F-cholesterol was reported to exhibit biological and interfacial properties similar to unmodified cholesterol. We have also found that 6-F-cholesterol mimicked the cholesterol activity observed in the systems of amphotericin B and lipid rafts. However, to use 6-F-cholesterol as a molecular probe to explore molecular recognition in membranes, it is indispensable to have detailed knowledge of the dynamic and orientation properties of the molecule in membrane environments. In this paper, we present the molecular orientation of 6-F-cholesterol (30 mol %) in dimyristoylphosphatidylcholine (DMPC) bilayers revealed by combined use of 19F chemical shift anisotropy (CSA), 2H NMR, and C-F rotational echo double resonance (REDOR) experiments. The axis of rotation of 6-F-cholesterol was shown to be in a similar direction to that of cholesterol in DMPC bilayers, which is almost parallel to the long axis of the molecular frame. The molecular order parameter of 6-F-cholesterol was determined to be ca. 0.85, which is within the range of reported values of cholesterol. These findings suggest that the dynamic properties of 6-F-cholesterol in DMPC are quite similar to those of unmodified cholesterol; therefore, the introduction of a fluorine atom at C6 has virtually no effect on cholesterol dynamics in membranes. In addition, this study demonstrates the practical utility of theoretical calculations for determining the 19F CSA principal axes, which would be extremely difficult to obtain experimentally. The combined use of quantum calculations and solid-state 19F NMR will make it possible to apply the orientation information of 19F CSA tensors to membrane systems..
76. Nagy Morsy, Toshihiro Houdai, Keiichi Konoki, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Effects of lipid constituents on membrane-permeabilizing activity of amphidinols, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2007.12.029, 16, 6, 3084-3090, 2008.03, Amphidinols (AMs) are a new class of polyhydroxyl polyene compounds with potent antifungal activity. Membrane-permeabilizing activities of AM2, AM3, and AM6 were examined using fluorescent-dye leakage experiments with various phosphatidylcholines (PCs) and sterols. All the AMs tested showed the potent activity to cholesterol-containing liposomes. In the absence of the sterol, AM2, AM3, and AM6 had no membrane-permeabilizing activities to membranes of saturated PC. In liposomes consisting of unsaturated PC, AM2, which possesses an additional ether ring in a polyhydroxyl chain, showed membrane-permeabilizing activities with a moderate efficacy, while AM3 or AM6 did not. The potentiation by sterols was prominent even at 0.5% (wt/wt) and structure-dependent, which ruled out the possibility that alteration of the membrane physical properties induced by sterol was chiefly responsible for this sterol effect. The finding that their activity was not affected by membrane thickness implies that AMs permeabilized membrane by a different mechanism from that of polyene macrolide antibiotics..
77. Yusuke Kasai, Nobuaki Matsumori, Yuichi Umegawa, Shigeru Matsuoka, Hiroyuki Ueno, Hiroki Ikeuchi, Tohru Oishi, Michio Murata, Self-assembled amphotericin B is probably surrounded by ergosterol
Bimolecular interactions as evidenced by solid-state NMR and CD spectra, Chemistry - A European Journal, 10.1002/chem.200701256, 14, 4, 1178-1185, 2008.02, Amphotericin B (AmB) is thought to exert its pharmacological effects by forming a barrel-stave assembly with ergosterol in fungal membranes. To examine the interaction between AmB and ergosterol (Erg) or cholesterol (Cho), 13C- and 19F-labelled covalent conjugales were prepared as reported previously (N. Matsumori et al. Chem. Biol. 2004, 11, 673-679). The CD spectra of the conjugates in a membrane-bound form suggested that the distance between the heptaene moieties of the ergosterol conjugates AmB-C 2-(6-F)Erg 2 and AmB-C2Erg 3 is similar to that of AmB in ergosterol-containing membranes, but significantly larger than that of AmB in nonsterol or cholesterol-containing mem branes. These observations suggest that, as is the case with ergosterol-containing membranes, the conjugated sterol moiety prevents the close contact between the heptaene moieties within the membrane that would reduce channel conductivity of the AmB assemblies. To further investigate this bimolecular interaction, we recorded the solid-state NMR spectra of conjugates 2 and AmB-C2-(6-F)Cho 4. which are composed of uniformly 13C-labelled AmB and 6-fluorinuted ergosterol or cholesterol; the conjugates were expected to facilitate the estimation of distances between the fluorine and carbon atoms. By using rotor-synchronous double resonance (rotational echo double resonance of X cluster; RDX) experiments, we deduced the distance between the fluorine atom and its nearest carbon atom in the heptaene moiety of 2 to be less than 8.6 Å. This indicates that the B ring of ergosterol comes close to the AmB polyene moiety. A conformational search of the AmB-ergosterol conjugate by using distance constraints derived from the RDX results suggested that ergosterol molecules possibly surround the AmB assembly, which is in contrast with the conventional image in which ergosterol is inserted into AmB molecules..
78. Nobuaki Matsumori, Atsushi Morooka, Michio Murata, Conformation and location of membrane-bound salinomycin-sodium complex deduced from NMR in isotropic bicelles, Journal of the American Chemical Society, 10.1021/ja075024l, 129, 48, 14989-14995, 2007.12, An ionophore antibiotic salinomycin was studied in a membrane environment consisting of isotropic bicelles, a better model for biological membranes than micelles, and its conformation and topological orientation in bicelles was determined. 2D NMR measurements and restrained conformational search revealed that salinomycin-sodium salt in bicelles adopts an open conformation in which the orientation of the E-ring is significantly different from that in crystal and solution structures. This conformational alteration breaks an intramolecular hydrogen bond between 28-OH and 1-O, dislocates the ether oxygen of the E-ring from a coordinated position to the sodium ion observed in the crystal, and consequently weakens the complexation between salinomycin and the sodium ion. Paramagnetic relaxation experiments using doxylphospholipids reveal that salinomycin is embedded shallowly in bicelles, with both terminals being closer to the water interface and the olefin portion facing the bicelle interior. Measurements of intermolecular NOEs between salinomycin and phospholipids further supported this orientation. Weaker complexation with sodium ion and positional preference in the membrane polar region may facilitate the catch-and-release of metal ions at the polar/nonpolar interface of bilayers. On the basis of these findings, a model for salinomycin-assisted transport of metal ions across biological membranes is proposed..
79. Yuichi Umegawa, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Amphotericin B covalent dimers with carbonyl-amino linkage
a new probe for investigating ion channel assemblies, Tetrahedron Letters, 10.1016/j.tetlet.2007.03.058, 48, 19, 3393-3396, 2007.05, Based on an amphotericin B (AmB) ion-channel model where the close proximity of neighboring molecules is effected by interaction between carboxyl and amino groups, we prepared covalent dimers of AmB connected between these functionalities. While directly connected and short-tethered derivatives (2 and 3) lacked the activities, dimer 4 with a longer linker revealed K+ ion flux activity, suggesting that some distance and/or flexibility between the carboxyl and amino groups in adjacent molecules is required for the formation of ion-permeable complex in biomembranes..
80. Shohei Sakuda, Nobuaki Matsumori, Kazuo Furihata, Hiromichi Nagasawa, Assignment of the absolute configuration of blasticidin A and revision of that of aflastatin A, Tetrahedron Letters, 10.1016/j.tetlet.2007.02.024, 48, 14, 2527-2531, 2007.04, The absolute configuration of blasticidin A, a strong inhibitor of aflatoxin production by Aspergillus parasiticus, was assigned by adding the data of relative configurations at its diol and pentaol moieties to previously known stereochemistry. Similarity of the NMR data of blasticidin A to those of aflastatin A allowed us to revise the stereochemistry of the diol and pentaol moieties of aflastatin A..
81. Nobuaki Matsumori, Toshihiro Houdai, Michio Murata, Conformation and position of membrane-bound amphotericin B deduced from NMR in SDS micelles, Journal of Organic Chemistry, 10.1021/jo061309p, 72, 3, 700-706, 2007.02, (Figure Presented) Amphotericin B (AmB) is known to self-assemble to form an ion channel across lipid bilayer membranes. To gain insight into the conformation of AmB in lipidic environments, AmB in SDS micelles was subjected to high-resolution NMR and CD measurements, and the NMR-derived conformation thus obtained was refined by molecular mechanics calculations. These results indicate that AmB in SDS micelles is conformationally fixed particularly for the macrolide moiety. Paramagnetic relaxation experiments with the use of Mn 2+ reveal that AmB is shallowly embedded in the micelle with the polyhydroxyl chain being close to the water interface and the side of polyene portion facing to the micelle interior. CD measurements demonstrate that AmB is in a monomeric form in SDS micelles. The structure of AmB in the micelles obtained in the present study may reproduce the initial stage of membrane interaction of AmB prior to the assembly formation in biomembranes..
82. Kohei Torikai, Hiroshi Yari, Megumi Mori, Satoru Ujihara, Nobuaki Matsumori, Michio Murata, Tohru Oishi, Design and synthesis of an artificial ladder-shaped polyether that interacts with glycophorin A, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2006.09.004, 16, 24, 6355-6359, 2006.12, Ladder-shaped polyether (LSP) compounds, such as brevetoxins and ciguatoxins, are thought to interact with transmembrane (TM) proteins. As a model LSP compound, we designed and synthesized an artificial tetracyclic ether (1) and evaluated its interaction with glycophorin A (GpA), a membrane protein known to dimerize or oligomerize between membrane-integral α-helical domains. Model compound 1 was found to induce the dissociation of oligomeric GpA in a similar manner to natural LSPs when examined by SDS-PAGE. The results suggest that even an artificial tetracyclic ether possesses the ability to interact with TM proteins, presumably through the intermolecular hydrogen bonds (Cα-H ⋯ O) with the GXXXG motif..
83. Shigeru Matsuoka, Hiroki Ikeuchi, Yuichi Umegawa, Nobuaki Matsumori, Michio Murata, Membrane interaction of amphotericin B as single-length assembly examined by solid state NMR for uniformly 13C-enriched agent, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2006.06.001, 14, 19, 6608-6614, 2006.10, The membrane interaction of amphotericin B (AmB), one of the most important anti-fungal drugs, was investigated by solid state NMR measurements of uniformly 13C-enriched AmB, which was prepared by the culture of the drug-producing microorganism in the presence of [u-13C6]glucose. All the 13C NMR signals of AmB upon binding to DLPC membrane were successfully assigned on the basis of the 13C-13C correlation spectrum. 13C-31P RDX (Rotational-Echo Double Resonance for X-clusters) experiments clearly revealed the REDOR dephasing effects for carbon atoms residing in the both terminal parts, whereas no dephasing was observed for the middle parts including polyolefinic C20-C33 and hydroxyl-bearing C8/C9 parts. These observations suggest that AmB binds to DLPC membrane with a high affinity to the phospholipid and spans the membrane with a single molecular length..
84. Nagy Morsy, Toshihiro Houdai, Shigeru Matsuoka, Nobuaki Matsumori, Seiji Adachi, Tohru Oishi, Michio Murata, Takashi Iwashita, Tsuyoshi Fujita, Structures of new amphidinols with truncated polyhydroxyl chain and their membrane-permeabilizing activities, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2006.06.012, 14, 19, 6548-6554, 2006.10, Two new homologues of amphidinols (AM14 and AM15) were isolated from the cultured dinoflagellate Amphidinium klebsii. The structures were elucidated on the basis of 2D NMR and collision-induced dissociation MS/MS and turned out to be closely related homologues of AM7. Their weak membrane-disrupting activity indicates that the hydrophobic polyene chain is essential for the potent biological activities. Structure-activity relationship for the polyhydroxyl part was then examined with use of AM homologues possessing various chain lengths, indicating that the pore size of the channel/lesion formed by AMs was not greatly affected by the length of the polyhydroxyl chain..
85. Nobuaki Matsumori, Yuri Sawada, Michio Murata, Large molecular assembly of amphotericin B formed in ergosterol-containing membrane evidenced by solid-state NMR of intramolecular bridged derivative, Journal of the American Chemical Society, 10.1021/ja063433w, 128, 36, 11977-11984, 2006.09, Amphotericin B (AmB 1) is known to assemble and form an ion channel across biomembranes. We have recently reported that conformation-restricted derivatives of AmB 2-4 show different ergosterol preferences in ion-channel assays, which suggested that the orientation of the mycosamine strongly affects the sterol selectivity of AmB. The data allowed us to assume that compound 3 showing the highest selectivity would reflect the active conformation of AmB in the channel assembly. In this study, to gain further insight into the active conformation of AmB, we prepared a new intramolecular-bridged derivative 5, where the linker encompassed a hydrophilic glycine moiety. The derivative has almost equivalent ion-channel activity to those of AmB and 3. The antifungal activity of 5 compared with 3 improves significantly, possibly because the increasing hydrophilicity in the linker enhances the penetrability through the fungal cell wall. Conformation of 5 was well converged and very similar to that of 3, thus further supporting the notion that the conformations of these derivatives reproduce the active structure of AmB in the channel complex. Then we used the derivative to probe the mobility of AmB in the membrane by solid-state NMR. To measure dipolar couplings and chemical shift anisotropies, we incorporated [1-13C,15N]glycine into the linker. The results indicate that 5 is mostly immobilized in ergosterol-containing DMPC bilayers, implying formation of large aggregates of 5. Meanwhile some fraction of 5 remains mobile in sterol-free DMPC bilayers, suggesting promotion of Amb aggregation by ergosterol..
86. Hiroshi Tsuchikawa, Naohiro Matsushita, Nobuaki Matsumori, Michio Murata, Tohru Oishi, Synthesis of 28-19F-amphotericin B methyl ester, Tetrahedron Letters, 10.1016/j.tetlet.2006.06.159, 47, 35, 6187-6191, 2006.08, A fluorinated amphotericin B (AmB) derivative, 28-19F-AmB methyl ester (3), labeled at the polyene moiety, was synthesized by combining chemical synthesis with degradation of a natural product via cross-coupling reactions and macrolactonization. The fluorinated derivative 3 showed antifungal activity similar to that of AmB, and is expected to be a powerful tool for NMR-based investigation of the mechanism of ion-channel formation..
87. Nobuaki Matsumori, Atsushi Morooka, Michio Murata, Detailed description of the conformation and location of membrane-bound erythromycin A using isotropic bicelles, Journal of Medicinal Chemistry, 10.1021/jm051210v, 49, 12, 3501-3508, 2006.06, Although many nonpeptidic drugs target biological membrane and membrane proteins, it is still difficult to define the membrane-bound structure of the drugs. In this study, we utilized bicelles as a membrane model, since the bicelles, which have planar lipid bilayer portions, are thought to be a more appropriate and practical membrane model than micelles. Bicelles with a small diameter allow for measurements of liquid NMR due to fast tumbling in solution. We targeted erythromycin A (EA) as a membrane-binding compound because it is pointed out that the drug interacts with lysosomal membranes, inhibits phospholipase A, and consequently induces phospholipidosis as a side effect. The conformation of EA in the bicelle was successfully determined on the basis of coupling constants and NOEs. Measurements of intermolecular NOEs and paramagnetic relaxation times revealed that the drug is located shallowly in the membrane surface, with the dimethylamino group being close to the phosphate, and the macrolide portion adjacent to upper sides of the acyl chains. This study shows the general utility of isotropic bicelles for detailed conformational and orientational studies of membrane-associated nonpeptidic drugs..
88. Nobuaki Matsumori, Tohru Oishi, Michio Murata, Derivatization and isotope labeling of amphotericin B aiming at elucidation of the ion-channel structure, Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry, 64, 5, 502-513, 2006.05, Amphotericin B (AmB) is known to assemble together and form an ion channel across biomembranes. The selective toxicity is generally accounted for by its higher affinity for ergosterol than cholesterol. To better understand the ion-channel structure and intermolecular interactions, we prepared various AmB derivatives and measured their activities. AmB dimers which are covalently linked between the amino groups by short chains showed more potent ion-channel activity than that of AmB, indicating that the mutual topology of AmB molecules is a head-to-head orientation. AmB-sterol conjugates were also designed and examined for ion-channel activities. AmB-ergosterol conjugates showed more powerful ion-channel activity than AmB-cholesterol congeners, suggesting that stronger van der Waals interaction between AmB and ergosterol contributes to the higher ion-channel activity. Finally, we prepared conformation-restricted derivatives of AmB, in which the amino and carboxyl groups were bridged with various lengths of alkyl chains. The derivatives successfully gave us information on the active-conformation of the sugar moiety of AmB in membrane. These derivatives are now labeled by 13C and/or 19F to probe the molecular structure of AmB ion channel using solid-state NMR..
89. Megumi Mori, Tohru Oishi, Shigeru Matsuoka, Satoru Ujihara, Nobuaki Matsumori, Michio Murata, Masayuki Satake, Yasukatsu Oshima, Nobuto Matsushita, Saburo Aimoto, Ladder-shaped polyether compound, desulfated yessotoxin, interacts with membrane-integral α-helix peptides, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2005.05.039, 13, 17, 5099-5103, 2005.09, Ladder-shaped polyether compounds, represented by brevetoxins, ciguatoxins, maitotoxin, and prymnesins, are thought to possess the high affinity to transmembrane proteins. As a model compound of ladder-shaped polyethers, we adopted desulfated yessotoxin (2) and examined its interaction with glycopholin A, a membrane protein known to form a dimer or oligomer. Desulfated yessotoxin turned out to interact with the α-helix so as to induce the dissociation of glycopholin oligomers when examined by SDS and PFO gel electrophoresis. The results provided the first evidence that lapper-shaped polyethers interact with transmembrane helix domains..
90. Nagy Morsy, Shigeru Matsuoka, Toshihiro Houdai, Nobuaki Matsumori, Seiji Adachi, Michio Murata, Takashi Iwashita, Tsuyoshi Fujita, Isolation and structure elucidation of a new amphidinol with a truncated polyhydroxyl chain from Amphidinium klebsii, Tetrahedron, 10.1016/j.tet.2005.07.004, 61, 36, 8606-8610, 2005.09, A new member of amphidinols (AM7) possessing a polyene-polyhydroxyl structure with the shortest carbon backbone and sulfate ester was isolated from the cultured dinoflagellate, Amphidinium klebsii. AM7 showed hemolytic and antifungal activities. The structure was elucidated on the basis of 2D NMR data in combination with CID MS/MS experiments..
91. Nobuaki Matsumori, Yuichi Umegawa, Tohru Oishi, Michio Murata, Bioactive fluorinated derivative of amphotericin B, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2005.05.058, 15, 15, 3565-3567, 2005.08, The first stably fluorinated derivative of amphotericin B (2) with a fluorine atom in the macrolide skeleton was synthesized using an electrophilic fluorination reagent, Selectfluor®. The derivative 2 showed hemolytic, K+ permeable, and antifungal activities similar to those of AmB and thus was expected to be a powerful tool for NMR-based investigations on the mechanism of ion-channel formation by amphotericin B..
92. Nobuaki Matsumori, Yuri Sawada, Michio Murata, Mycosamine orientation of amphotericin B controlling interaction with ergosterol
Sterol-dependent activity of conformation-restricted derivatives with an amino-carbonyl bridge, Journal of the American Chemical Society, 10.1021/ja051597r, 127, 30, 10667-10675, 2005.08, Amphotericin B (AmB 1) is known to assemble together and form an ion channel across biomembranes. The antibiotic consists of mycosamine and macrolactone moieties, whose relative geometry is speculated to be determinant for the drug's channel activity and sterol selectivity. To better understand the relationship between the amino-sugar orientation and drug's activity, we prepared conformation-restricted derivatives 2-4, in which the amino and carboxyl groups were bridged together with various lengths of alkyl chains. K+ influx assays across the lipid-bilayer membrane revealed that ergosterol selectivity was markedly different among derivatives; short-bridged derivative 2 almost lost the selectivity, while 3 showed higher ergosterol preference than AmB itself. Monte Carlo conformational analysis of 2-4 based on NOE-derived distances indicated that the amino-sugar moiety of 2 comes close to C41 because of the short bridge, whereas those of 3 and 4 are pointing outward. The mutual orientation of the amino-sugar moiety and macrolide ring is so rigid in derivatives 2 and 3 that these conformations should be unchanged upon complex formation in lipid membranes. These results strongly suggest that the large difference in sterol preference between derivatives 2 and 3 is ascribed to the different orientation of amino-sugar moieties. These findings allowed us to propose a simple model accounting for AmB-sterol interactions, in which hydrogen bonding between 2′-OH of AmB and 3β-OH of ergosterol plays an important role..
93. Toshihiro Houdai, Shigeru Matsuoka, Nagy Morsy, Nobuaki Matsumori, Masayuki Satake, Michio Murata, Hairpin conformation of amphidinols possibly accounting for potent membrane permeabilizing activities, Tetrahedron, 10.1016/j.tet.2005.01.069, 61, 11, 2795-2802, 2005.03, Amphidinols are a unique dinoflagellate metabolite with potent antifungal activity. We examined membrane permeabilizing action by amphidinol analogues with structural variations in polyhydroxy and polyene moieties. Consequently, the polyene and polyhydroxy moieties turned out to play important roles in binding to lipid bilayer membrane and in forming ion-permeable pore/lesion across membrane, respectively. NMR-constrained modeling experiments have revealed for the fist time that amphidinols in membrane generally take a hairpin configuration, which plausibly accounts for their potent antifungal and other membrane permeabilizing activities..
94. Shigeru Matsuoka, Hiroki Ikeuchi, Nobuaki Matsumori, Michio Murata, Dominant formation of a single-length channel by amphotericin B in dimyristoylphosphatidylcholine membrane evidenced by 13C- 31P rotational echo double resonance, Biochemistry, 10.1021/bi049001k, 44, 2, 704-710, 2005.01, 13C-Labeled amphotericin B (AmB) was prepared by feeding the producing organism Streptomyces nodosus with [3-13C]propionate. The REDOR experiments for dimyristoylphosphatidylcholine (DMPC) membrane using the 13C-labeled AmB showed the prominent dephasing effects between the phosphate group in PC and C41 carboxyl carbon in the polar head. In addition, C39/C40 methyl carbons also gave rise to the significant reduction of their 13C NMR signals, implying that both terminal parts of AmB reside close to the surface of the DMPC membrane. Conversely, the same REDOR experiments with use of distearoylphosphatidylcholine (DSPC) showed no dephasing for the C39/C40 methyl signals while a marked reduction of the C41 carbonyl signal was again observed. These findings should be most reasonably accounted for by the notion that AmB can span across the DMPC membrane with a single-length interaction but cannot span the DSPC membrane due to its greater thickness. To our knowledge, the results provide the first direct spectroscopic evidence for the formation of a single-length channel across a biomembrane, which was previously suggested by channel current recording experiments..
95. Nobuaki Matsumori, Rie Masuda, Michio Murata, Amphotericin B covalent dimers bearing a tartarate linkage, Chemistry and Biodiversity, 10.1002/cbdv.200490030, 1, 2, 346-352, 2004.12, Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 μm) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB..
96. Toshihiro Houdai, Shigeru Matsuoka, Nobuaki Matsumori, Michio Murata, Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate, Biochimica et Biophysica Acta - Biomembranes, 10.1016/j.bbamem.2004.09.002, 1667, 1, 91-100, 2004.11, Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, 23Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na + flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism..
97. Keisuke Maruyoshi, Tetsuo Demura, Takeshi Sagane, Nobuaki Matsumori, Tohru Oishi, Michio Murata, Synthesis and conformation of deuterated spermidine for investigating weak interaction with polyanionic biomolecules, Tetrahedron, 10.1016/j.tet.2004.04.056, 60, 24, 5163-5170, 2004.06, Polyamines such as spermidine and spermine are known to interact with polyanionic biomolecules under physiological conditions. Conformation analysis of these highly flexible acycles is virtually unprecedented due to the lack of appropriate methodologies. Spin-spin coupling constants, which are often utilized for acyclic systems, are rather uninformative for polyamines due to unresolved 1H NMR signals of their tri- and tetra-methylene moieties. We attempted to solve this problem by synthesizing diastereospecifically deuterated spermidine, (1S*,2S*)-1,8-diamino- 4-azaoctane-1,2,3,3,5,5,6,6,7,7,8,8-d12. To evaluate its utility, 1H-1H spin coupling constants were measured as trihydrochloride or tripolyphosphate salt. The relevant coupling constant markedly decreased upon complexation to tripolyphosphate. Under neutral pH, where the net charge of tripolyphosphate became tetravalent, the coupling constant was small; as pH was lowered to 1.4, where the charge was divalent, the constant increased significantly. These data clearly indicate that, when interacting with polyanions, spermidine undergoes conformational changes to increase bent conformation, which could be effectively monitored by the deuterated spermidine..
98. Nobuaki Matsumori, Noritsugu Eiraku, Shigeru Matsuoka, Tohru Oishi, Michio Murata, Takaaki Aoki, Toru Ide, An amphotericin B-ergosterol covalent conjugate with powerful membrane permeabilizing activity, Chemistry and Biology, 10.1016/j.chembiol.2004.02.027, 11, 5, 673-679, 2004.05, Amphotericin B-sterol conjugates were synthesized and examined for their membrane permeabilizing activity. Ergosterol and cholesterol, each connected with amphotericin B via an ethylenecarbamate or hexamethylenecarbamate linker, were examined by K+ flux assays using liposomes and by single-channel recording across phospholipid membrane. Among four conjugates tested, AmB-ergosterol bearing an ethylenecarbamate linker exhibited the most powerful activity, which substantially exceeded that of the cholesterol homolog. Single-channel recording clearly exhibited that the ergosterol conjugate elicited channel current with the conductance of 28 pS, which was comparable with those by AmB, and revealed a higher channel open probability than the cholesterol conjugate. These results imply that direct interaction between amphotericin B and ergosterol is reproduced by their conjugate, which may serve as a model compound for understanding the drug's selective toxicity..
99. Shigeru Matsuoka, Nobuaki Matsumori, Michio Murata, Amphotericin B-phospholipid covalent conjugates
Dependence of membrane-permeabilizing activity on acyl-chain length, Organic and Biomolecular Chemistry, 10.1039/b306801c, 1, 22, 3882-3884, 2003.11, The interaction between amphotericin B and phospholipid upon forming ion channels across a biomembrane was investigated using their covalent conjugates. The membrane permeabilizing activity was greatly affected by the chain length of the fatty acyl groups, suggesting that their interaction is involved in ion channel assemblages..
100. Nahoko Yamaji, Nobuaki Matsumori, Shigeru Matsuoka, Tohru Oishi, Michio Murata, Amphotericin B Dimers with Bisamide Linkage Bearing Powerful Membrane-Permeabilizing Activity, Organic Letters, 10.1021/ol025982m, 4, 12, 2087-2089, 2002.06, (Matrix Presented) Covalently linked dimers of amphotericin B were prepared by cross-linking its carboxylic acid. Among these, a dimer with a linkage of 1,6-hexanediamine revealed potent hemolytic activity (EC50,0.25 μM) while its N-acetyl derivative gave rise to large K+ ion flux in phosphatidylcholine liposomes, regardless of the presence or absence of sterols, suggesting that the dimers may serve as a tool for elucidating the structure of the ion channel assemblage formed by amphotericin B..
101. Nobuaki Matsumori, Nahoko Yamaji, Shigeru Matsuoka, Tohru Oishi, Michio Murata, Amphotericin B covalent dimers forming sterol-dependent ion-permeable membrane channels, Journal of the American Chemical Society, 10.1021/ja012026b, 124, 16, 4180-4181, 2002.04, Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for a long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K+ influx actions monitored by measuring the pH of the liposome lumen by 31P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in 31P NMR spectra, the dimers appeared to induce "all-or-none"-type ion flux across the liposome membrane in the presence of ergosterol, which suggested that the ion channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements..
102. Hiroyuki Ikeda, Nobuaki Matsumori, Makoto Ono, Akinori Suzuki, Akira Isogai, Hiromichi Nagasawa, Shohei Sakuda, Absolute configuration of aflastatin A, a specific inhibitor of aflatoxin production by Aspergillus parasiticus, Journal of Organic Chemistry, 10.1021/jo991284c, 65, 2, 438-444, 2000.01, Aflastatin A (1) is a specific inhibitor of aflatoxin production by Aspergillus parasiticus. It has the novel structure of a tetramic acid derivative with a long alkyl side chain. The absolute configurations of 29 chiral centers contained in I were chemically elucidated in this study. First, four small fragment molecules were prepared from I or its methyl ether (2), and their absolute structures were assigned as N-methyl-D-alanine, (2S,4R)-2,4-dimethyl-1,6-hexanediol dibenzoate, (R)-3-hydroxydodecanoic acid, and (R)-1,2,4-butanetriol tribenzoate. Next, an acyclic fragment molecule 3 with 13 chiral centers was obtained from I by NaIO4 oxidation, and its relative stereochemistry was elucidated by J-based configuration analysis. By analyzing coupling constants of 3J(H,H) and 2,3J(C,H) and ROE data, the relative configuration of 3 was verified. Finally, by further J-based configuration analysis using a fragment molecule 7 prepared from 2 with 28 chiral carbons, all relative configurations in the alkyl side chain of I were clarified. By connecting these relative configurations with the absolute configurations of first four fragment molecules, the absolute stereochemistry of 1 was fully determined..
103. Nobuaki Kudo, Nobuaki Matsumori, Hiroshi Taoka, Daisuke Fujiwara, Erwin P. Schreiner, Barbara Wolff, Minoru Yoshida, Sueharu Horinouchi, Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.96.16.9112, 96, 16, 9112-9117, 1999.08, The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizo-saccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB. 1H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its α,β-unsaturated δ-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N- ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation..
104. Michio Murata, Shigeru Matsuoka, Nobuaki Matsumori, Gopal K. Paul, Kazuo Tachibana, Absolute configuration of amphidinol 3, the first complete structure determination from amphidinol homologues
Application of a new configuration analysis based on carbon-hydrogen spin-coupling constants [8], Journal of the American Chemical Society, 10.1021/ja983655x, 121, 4, 870-871, 1999.02.
105. Nobuaki Matsumori, Daisuke Kaneno, Michio Murata, Hideshi Nakamura, Kazuo Tachibana, Stereochemical determination of acyclic structures based on carbon- proton spin-coupling constants. A method of configuration analysis for natural products, Journal of Organic Chemistry, 10.1021/jo981810k, 64, 3, 866-876, 1999.02, A method for elucidating the relative configuration of acyclic organic compounds was developed on the basis of carbon-proton spin-coupling constants (2,3J(C,H)) and interproton spin-coupling constants (3J(H,H)). This method is based on the theory that, in acyclic systems, the conformation of adjacent asymmetric centers is represented by staggered rotamers, and their relative stereochemistry can be determined using 2,3J(C,H) and 3J(H,H), because the combined use of these J values enables the identification of the predominant staggered rotamer(s) out of the six possible derived from threo and erythro configurations. Detailed conformational analysis for model compounds 1-4 revealed that this method is useful in most cases for assignment of the configuration of acyclic structures occurring in natural products, in which stereogenic methine carbons are often substituted with a methyl or a hydroxy (alkoxy) group. This J-based configuration analysis was applied to the stereochemical elucidation of carboxylic acid 5 derived from zooxanthellatoxin and proven to be a practical method even for natural products with complicated structures..
106. Hiroyuki Onuki, Kei Ito, Yoshimasa Kobayashi, Nobuaki Matsumori, Kazuo Tachibana, Nobuhiro Fusetani, Absolute structure and total synthesis of lipogrammistin-A, a lipophilic ichthyotoxin of the soapfish, Journal of Organic Chemistry, 10.1021/jo9722461, 63, 12, 3925-3932, 1998.06, Lipogrammistin-A (1b) was isolated as an ichthyotoxic and hemolytic constituent of the skin mucus of the grammistid fish Aulacocephalus temmincki. Its absolute stereochemistry was established by chemical degradation and total synthesis. The stereochemistry of the 2-methylbutyryl moieties attached to N9 and N15 was determined to be S by HPLC analysis of a diastereomeric derivative. The stereochemistry of the remaining C4 position was elucidated to be S by a total synthesis of the two diastereomers (4S)- and (4R)-1..
107. Michio Murata, Nobuaki Matsumori, Kazuo Tachibana, Analysis of relative configuration of acyclic compounds based on long-range carbon-proton coupling constants determined by two dimensional NMR, Unknown Journal, 10.1246/nikkashi.1997.749, 11, 749-757, 1997.01, A new NMR methodology was devised for configurational assignments of acyclic structures in natural products or synthetic compounds. Conventional methods based on NOEs often suffer from the ambiguity in assigning the configurations of compounds with acyclic structures and conformational alternation. Unlike these NOE analyses, the new methodology is based on the dihedral angle dependency of long-range carbon-proton coupling constants (2,3JC.H). The combination of the coupling constants (2,3JC.H, 3JH,H) which are categorized to large, medium, or small allows to determine the relative stereochemistry among adjacent asymmetric centers or those separated by a methylene group. Hetero half-filter TOCSY (HETLOC) was shown to be the most effective for measuring 2,3JC,H of protonated carbon system. For structures with quaternary carbons or weakly coupling protons, in which TOCSY in HETLOC fails to correlate relevant protons, phase-sensitive HMBC (HMQC optimized for long-range coupling) worked effectively. To evaluate the utility of the method in structural analysis of natural products, the relative configurations of acyclic portions of maitotoxin, the largest secondary metabolite known to date, were assigned by this method, which evidently demonstrated the validity of 2,3JC,H in the configurational and conformational analysis of organic compounds..
108. Noriko Ando, Nobuaki Matsumori, Shohei Sakuda, Teruhiko Beppu, Sueharu Horinouchi, Involvement of AfsA in A-factor biosynthesis as a key enzyme, Journal of Antibiotics, 10.7164/antibiotics.50.847, 50, 10, 847-852, 1997.01, The afsA gene of Streptomyces griseus has been postulated to encode a key enzyme for A-factor biosynthesis from primary metabolites commonly present in Streptomyces strains. Escherichia coli cells harboring afsA under the control of the T7 promoter specified distinct A-factor activity in the culture broth, as determined by induction of streptomycin production and aerial mycelium and spore formation in an A-factor-deficient S. griseus mutant strain. Production of the substance(s) having A-factor activity was inhibited by cerulenin, an inhibitor of fatty acid biosynthesis. These observations suggest that afsA encodes a key enzyme in the A-factor biosynthetic pathway in which a β-keto acid derived from fatty acid biosynthesis and a glycerol derivative serve as precursors..
109. Gopal K. Paul, Nobuaki Matsumori, Keiichi Konoki, Michio Murata, Kazuo Tachibana, Chemical structures of amphidinols 5 and 6 isolated from marine dinoflagellate Amphidinium klebsii and their cholesterol-dependent membrane disruption, Journal of Marine Biotechnology, 5, 2-3, 124-128, 1997, A series of polyhydroxy-polyene compounds were isolated from the cultured dinoflagellate Amphidinium klebsii as well as from its surrounding media. Amphidinol 5 (AM 5) and 6 (AM 6) were found to be potent antifungal and hemolytic agents. Their permeabilizing activity towards phospholipid liposomes was markedly enhanced by the presence of cholesterol within the bilayer, indicating that the mechanism of action involved interaction with membranal cholesterol..
110. Nobuaki Matsumori, Taro Nonomura, Makoto Sasaki, Michio Murata, Kazuo Tachibana, Masayuki Satake, Takeshi Yasumoto, Long-range carbon-proton coupling constants for stereochemical assignment of acyclic structures in natural products
Configuration of the C5-C9 portion of maitotoxin, Tetrahedron Letters, 10.1016/0040-4039(95)02413-1, 37, 8, 1269-1272, 1996.02, Long-range carbon-proton coupling constants (2,3J(C,H)) were measured for maitotoxin (MTX), one of the largest natural non-biopolymers, by hetero-half filter experiments and phase-sensitive HMBC with use of 9 mg of a 4% 13C-enriched sample. The necessary coupling constants within the terminal acyclic portions of MTX, where NOE analysis was not successful owing to the presumed coexistence of multiple conformers, were thus obtained for the resultant elucidation of relative configurations for the acyclic stereogenic centers to the 5R*, 7R*, 8R*, and 9S*..
111. Taro Nonomura, Makoto Sasaki, Nobuaki Matsumori, Michio Murata, Kazuo Tachibana, Takeshi Yasumoto, The Complete Structure of Maitotoxin, Part II
Configuration of the C135-C142 Side Chain and Absolute Configuration of the Entire Molecule, Angewandte Chemie (International Edition in English), 10.1002/anie.199616751, 35, 15, 1675-1678, 1996.01.
112. Makoto Sasaki, Nobuaki Matsumori, Takahiro Maruyama, Taro Nonomura, Michio Murata, Kazuo Tachibana, Takeshi Yasumoto, The Complete Structure of Maitotoxin, Part I
Configuration of the C1-C14 Side Chain, Angewandte Chemie (International Edition in English), 10.1002/anie.199616721, 35, 15, 1672-1675, 1996.01.
113. Makoto Sasaki, Nobuaki Matsumori, Michio Murata, Kazuo Tachibana, Takeshi Yasumoto, Stereochemical assignment of the C35-C39 Acyclic linkage in maitotoxin
completion of stereochemical determination of C15-C134, Tetrahedron Letters, 10.1016/0040-4039(95)01973-L, 36, 49, 9011-9014, 1995.12, The relative configuration of carbons within the C35-C39 acyclic linkage of maitotoxin (MTX) was assigned by synthesis of stereodefined model compound 1 and its comparison with MTX in 1H and 13C NMR spectra. This result completed Stereochemical assignments of the whole molecule except for side chains..
114. Nobuaki Matsumori, Michio Murata, Kazuo Tachibana, Conformational analysis of natural products using long-range carbon-proton coupling constants
Three-dimensional structure of okadaic acid in solution, Tetrahedron, 10.1016/0040-4020(95)00790-F, 51, 45, 12229-12238, 1995.11, Long-range carbon-proton coupling constants (2,3JC,H) are known to depend on the dihedral angles as is the well-known case with interproton couplings. The precise measurement of these J values were carried out using hetero half-filtered TOCSY and phase-sensitive HMBC on a marine natural product, okadaic acid. From the obtained values together with 3JH,H we disclosed the conformations of okadaic acid in organic and aqueous solutions (CD3OD-CDCl3 or NaOD/D2O), which resembled each other and that obtained for its crystalline o-bromobenzyl ester. The successful result in the present conformational study, even though as an exmaple, demonstrated this methodology to be powerful in elucidation of conformations and configurations of acyclic or macrocyclic portions in natural products of this size..
115. Gopal K. Paul, Nobuaki Matsumori, Michio Murata, Kazuo Tachibana, Isolation and chemical structure of amphidinol 2, a potent hemolytic compound from marine dinoflagellate Amphidinium klebsii, Tetrahedron Letters, 10.1016/0040-4039(95)01259-K, 36, 35, 6279-6282, 1995.08, A new polyene-polyhydroxy compound, amphidinol 2 (2), was isolated as a potent hemolytic and antifungal agent from a cultured strain of dinoflagellate Amphidinium klebsii. The structure elucidated by spectroscopic methods turned out to be partly analogous to amphidinol 1 (1), which had been reported from the different strain of A. klebsii..