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ABSTRACT: 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.
Journal of Natural Products 11/2012; · 3.13 Impact Factor
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ABSTRACT: Lipid rafts have attracted much attention because of their significant functional roles in membrane-associated processes. It is thought that sphingomyelin and cholesterol are essential for forming lipid rafts; however, their motion characteristics are not fully understood despite numerous studies. Here we show accurate local motions encompassing an entire sphingomyelin molecule, which were captured by measuring quadrupole splittings for 19 kinds of site-specifically deuterated sphingomyelins (that is, molecular motion capture of sphingomyelin). The quadrupole splitting profiles, which are distinct from those reported from perdeuterated sphingomyelins or simulation studies, reveal that cholesterol enhances the order in the middle parts of the alkyl chains more efficaciously than at the shallow positions. Comparison with dimyristoylphosphocholine bilayers suggests that cholesterol is deeper in sphingomyelin bilayers, which likely explains the so-called umbrella effect. The experiments also demonstrate that (i) the C2'-C3' bond predominantly takes the gauche conformation, (ii) the net ordering effect of cholesterol in sphingomyelin bilayers is not larger than that in phosphatidylcholine bilayers, (iii) cholesterol has no specific preference for the acyl or sphingosine chain, (iv) the acyl and sphingosine chains seem mismatched by about two methylene lengths, and (v) the motion of the upper regions of sphingomyelin chains is less temperature dependent than that of lower regions probably due to intermolecular hydrogen bond formation among SM molecules. These insights into the atomic-level dynamics of sphingomyelin provide critical clues to understanding the mechanism of raft formation.
Biochemistry 09/2012; 51(42):8363-70. · 3.42 Impact Factor
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ABSTRACT: Aiming for structural analysis of amphotericin B (AmB) ion-channel assemblies in membrane, a covalent dimer was synthesized between (13)C-labled AmB methyl ester and (19)F-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 (13)C{(19)F}REDOR (Rotational-Echo Double Resonance) experiments in hydrated lipid bilayers. The obtained REDOR dephasing effects were explained by two components; a short (13)C/(19)F distance (6.9Å) accounting for 23% of the REDOR dephasing, and a longer one (14Å) comprising the rest of the dephasing. The shorter distance is likely to reflect the formation of barrel-stave ion channel.
Bioorganic & medicinal chemistry 08/2012; 20(19):5699-704. · 2.82 Impact Factor
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ABSTRACT: 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.
Bioorganic & medicinal chemistry 05/2012; 20(13):4012-9. · 2.82 Impact Factor
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ABSTRACT: 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 Ca(2+) 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 Ca(2+) influx that has ever been reported.
Bioorganic & medicinal chemistry letters 04/2012; 22(11):3619-22. · 2.65 Impact Factor
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ABSTRACT: 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 (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-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, (13)C-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, (13)C{(31)P}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.
Biochemistry 11/2011; 51(1):83-9. · 3.42 Impact Factor
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ABSTRACT: 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 (31)P NMR, (2)H 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 (1)H NMR and found that the C2-C6 and amide groups of SM take a relatively rigid conformation in bicelles.
Bioorganic & medicinal chemistry 11/2011; 20(1):270-8. · 2.82 Impact Factor
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ABSTRACT: 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.
Chemistry 07/2011; 17(31):8568-75. · 5.93 Impact Factor
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ABSTRACT: 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 (2)H 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.
Chemistry and physics of lipids 06/2011; 164(5):401-8. · 2.15 Impact Factor
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ABSTRACT: 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 (19)F 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 TiF(4) and n-Bu(4)N(+)Ph(3)SiF(2)(-). 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.
Organic & Biomolecular Chemistry 03/2011; 9(5):1437-42. · 3.70 Impact Factor
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ABSTRACT: Selectively (2)H- and (13)C-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-Mg(2+) , 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-Mg(2+) than with that of SPD, which chiefly interacts with β- and γ-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.
Chemistry 03/2011; 17(17):4788-95. · 5.93 Impact Factor
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ABSTRACT: 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-NH(2) (4) and Ac-(Pro-Hyp-Gly)(11)-Ala-NH(2) (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.
Bioorganic & medicinal chemistry 03/2011; 19(7):2125-9. · 2.82 Impact Factor
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ABSTRACT: 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.
Journal of Membrane Biology 03/2011; 240(3):159-64. · 1.81 Impact Factor
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Natural Product Reports 10/2010; 27(10):1480-92. · 9.79 Impact Factor
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Satoru Ujihara,
Tohru Oishi,
Ryota Mouri,
Rie Tamate,
Keiichi Konoki, Nobuaki Matsumori,
Michio Murata,
Yasukatsu Oshima,
Naoyuki Sugiyama,
Masaru Tomita,
Yasushi Ishihama
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ABSTRACT: 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 K(D) value around 4 μM.
Bioorganic & medicinal chemistry letters 09/2010; 20(22):6443-6. · 2.65 Impact Factor
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ABSTRACT: 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.
Bioorganic & medicinal chemistry letters 02/2010; 20(7):2215-8. · 2.65 Impact Factor
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ABSTRACT: 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 (2)H NMR using deuterium-labeled sterols and AmB. The (2)H 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.
Journal of the American Chemical Society 08/2009; 131(33):11855-60. · 9.91 Impact Factor
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ABSTRACT: 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.
Bioorganic & medicinal chemistry 08/2009; 17(17):6301-4. · 2.82 Impact Factor
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ABSTRACT: 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.
Bioorganic & medicinal chemistry letters 04/2009; 19(10):2824-8. · 2.65 Impact Factor
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ABSTRACT: 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. (1)H-(1)H and (13)C-(1)H 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.
Chemistry 02/2009; 15(7):1618-26. · 5.93 Impact Factor
