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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.

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.

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder arising from a single hematopoietic cell. Approximately 5% of reported cases of AMM have terminated in leukemic crisis; however, the precise characteristics of the leukemic cells have rarely been reported. We report a case of AMM that occurred in a 42-year-old man and was complicated by leukemic transformation. The leukemic cells were morphologically lymphoblastoid cells with a negative reaction to peroxidase staining, and phenotypically characterized as CD7+, CD34+, HLA-DR+, CD4-, CD8-, CD10-, CD13-, and CD33-. Southern blot analysis revealed that T cell receptor-beta, gamma, and immunoglobulin heavy chain genes in leukemic cells were retained in germ-line configuration. These observations suggest that leukemic cells in our case involved early hematopoietic stem cells rather than those strictly committed to myeloid or lymphoid precursors. To our knowledge, this is the first report of stem cell leukemia arising in a patient with AMM.

Defect levels in undoped n-InGaAs caused by the implantation of boron ions and their behaviour upon post-implantation rapid thermal annealing (RTA) have been investigated. Acceptorlike electron traps, which decreased electron density, which first formed by implantation at ∼ 0.2 eV below the conduction band edge. After furtherimplantation shallow donor states, which increased electron density, appeared, and limited the maximum attainable resistivity of InGaAs to several tens of ω cm. Post-implantation RTA at 600°C reduced the electron traps and activated further the shallow donors, leading to an increase in electron density to much higher values than that of the as-grown undoped InGaAs.

The structure of a new ciguatoxin congener, CTX3C (1). isolated from cultures of a dinoflagellate Gambierdiscus toxicus was elucidated mainly o

We have examined the applicability of tandem mass spectrometry to the structural elucidation of polyether compounds. The polyether toxin named yessotoxin was isolated from scallops which were implicated in food poisoning. The toxin consists of fused polycyclic ethers as does ciguatoxin, and bears two sulfate esters in the molecule. The negative-ion fast-atom bombardment tandem mass spectrum of the toxin showed a series of ions due to fragmentation at positions characteristic of cyclic ethers. With the mass spectrometric data alone, we succeeded in assigning the number and location of eleven ether rings in yessotoxin, which agreed with those previously deduced by nuclear magnetic resonance studies.

The frequency of streptococcal infections has been reported to be increasing. To determine the significance of such infections complicating treatment of leukemia, we studied the incidence and clinical features of Streptococcus mitis septicemia among 51 leukemia patients in our department. During 166 consecutive treatment courses for leukemia, eight episodes of Streptococcus mitis septicemia were observed in 35 septicemic patients. In seven out of eight episodes (88%), severe mucositis developed after aggressive chemotherapy, suggesting that oral mucosa might be the site of entry for Streptococcus mitis. The isolates were sensitive to imipenam/cilastatin and cefuzonam, and were relatively resistant to amikacin. Although none of the patients died of Streptococcus mitis septicemia, life-threatening adult respiratory distress syndrome (ARDS) developed in two independent treatment courses. We should thus be aware of the risk of ARDS in patients with Streptococcus mitis septicemia.

Covering: up to the end of 2009 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.

The formation of these two different complexes requires that the macrocycle adopt two opposite conformations in the binding of 2 and 3 (Figure 1). In forming a complex, two opposed nine-base domains move inward and align themselves with the complementary purine strand on opposite sides, forming two or three hydrogen bonds on each side of a base. The unbound nine-base domains then act as loops for bridging the bound domains. In order to bind the other sequence, the pairs of domains reverse roles, with the former binding domains acting as bridging loops and vice versa. Interestingly, a further mixing experiment reveals that this complexation is mutually exclusive: the act of binding to one sequence precludes the binding of the other, even though the necessary bases are unpaired in the loops. When the circle is mixed at varied molar ratios with a one-to-one mixture of the two sequences (Figure 2), the mixing plot shows a stoichiometry of 1:l. This indicates that a given complex cannot bind the second sequence with its loops at 25 OC. While this macrocycle at all times carries the bases necessary for forming complexes with the two sequences, the act of binding causes a conformational switch, holding the loops in an inaccessible or unproductive conformation.I0 To confirm that the macrocycle can recognize these two sequences within a longer strand of DNA, we synthesized a 33nucleotide oligomer having the sequence 5'dCACAAGAGAGAGAATCCCTAAAAAAAAAAACAC (4). This oligomer contains the two recognition sequences separated by seven bases. When mixing experiments are performed (pH 7.0, 25 "C) with this oligomer and the macrocycle 1, a plot of (7) Thermal denaturations were performed on a Cary 1 spectrophotometer in 1 cm path length stoppered quartz microcells under an N2 atmosphere. The cells were monitored at 260 nm, with a temperature rise of 0.5 OC/min. Mixtures were 3 pM in each DNA strand and contained 10 mM Na-PIPES buffer (pH 7.0), 100 mM NaCI, and 10 mM MgCI,. T,'s were taken as the inflection point in the denaturation curve and are estimated to be accurate to within 10.5 OC.

Stereocontrolled synthesis of cis-fused 1,6-dioxadecalin system 1, which corresponds to the L/M and N/O rings of maitotoxin, was accomplished. Comparison of its 1H and 13C NMR data with those of the natural toxin established the earlier stereochemical assignments.

The relative configuration of carbons within the C63–C68 acyclic linkage of maitotoxin (MTX) was assigned by synthesis of stereodefined model compounds ( 1a - 1d ) and their comparison with MTX in 1 H and 13 C NMR spectra.

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.

Long-range carbon-proton coupling constants ( 2,3 J C,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 3 J H,H we disclosed the conformations of okadaic acid in organic and aqueous solutions (CD 3 OD-CDCl 3 or NaOD/D 2 O), 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.

Maitotoxin (MTX, 1) is the most toxic and largest non-biopolymer known to date. The complete structural determination of MTX including the absolute configuration has been achieved recently. The relative configurations of the four acyclic portions (C1-C15, C35-C39, C63-C68, and C134-C142) were elucidated by (1) the extensive configurational analysis mainly using long-range carbon-proton coupling constants (2, 3JC, H), and (2) the stereoselective synthesis of the eight model compounds (2A-2D, 13, 27A, 27B, and 47) which were furnished with thus assigned relative configurations. The comparison of their 1H and 13C-NMR data with those for the corresponding portion of MTX evidently established the relative configuration from C1 to C138. The absolute configuration of the entire molecule was determined by the enantioselective synthesis and chiral gas chromatographic comparison with a degradated fragment (3, 4-dimethy1-6-hepten-1-o1) derived from the natural product, which involved two chiral centers at C138 and C139.The present results have revealed that the preferred conformation of an appropriately designed model compound with the correct configuration closely mimics that of the corresponding portion of MTX. Thus, we could deduce the overall conformation of MTX by assembling the preferred conformation of each model compound based on the NMR data.

Reductive etherification, a key reaction for constructing the H-ring in the convergent synthesis of the FGHIJ-ring system of yessotoxin, was problematic because the yield dramatically decreased upo...

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.

Gronbland—Strandberg syndrome is an inherited connective tissue disorder with cutaneous, opthalomologic, and systemic manifestations. Here we report a case of AML with thrombocytosis arising in a patient with this syndrome. Karyotypic analysis of bone marrow cells revealed the inversion 3 (q21.3;q26.2). To our knowledge, this is the first report of the association of AML with thrombocytosis and Gronbland—Strandberg syndrome. This might be a coincidental association, but it suggests some interesting speculations regarding the pathogenesis of these diseases.

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.

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.

The AB ring framework of gambiertoxin 4B which has a butadienyl substituent on the tetrahydrooxepin ring A has been synthesized stereoselectively and its CD spectrum suggests that gambiertoxin 4B and ciguatoxin have 5R configuration.

Lasing action of a surface-emitting laser with a two-dimensional photonic crystal structure is investigated. The photonic crystal has a triangular-lattice structure composed of InP and air holes, which is integrated with an InGaAsP/InP multiple-quantum-well active layer by a wafer fusion technique. Uniform two-dimensional lasing oscillation based on the coupling of light propagating in six equivalent Γ−X directions is successfully observed, where the wavelength of the active layer is designed to match the folded (second-order) Γ point of the Γ−X direction. The very narrow divergence angle of far field pattern and/or the lasing spectrum, which is considered to reflect the two-dimensional stop band, also indicate that the lasing oscillation occurs coherently.

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 (13)C{(19)F}REDOR experiments using 14-(19)F- and (13)C41-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.

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.

Ladder-shaped polyether (LSP) compounds are thought to interact with transmembrane alpha-helices, but direct evidence has scarcely obtained for these interactions. We adopted a transmembrane alpha-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 (K(D)) 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 alpha-helix. These results suggest that hydrophobic interaction plays an important role in molecular recognition of the alpha-helix peptide by LSPs.

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.

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.

The histopathological response of male ICR mice to yessotoxin, isolated from the digestive organ of scallops Patinopecten yessoensis , was compared with that of desulfated yessotoxin. The target organ of the former was the heart and those of the latter were the liver and pancreas. Electron microscopically, marked intracytoplasmic edema in cardiac muscle cells was seen within 3 hr after the i.p. injection of over 300 μg/kg of yessotoxin. In contrast, desulfated yessotoxin at the same dose caused within 24 hr of i.p. injection severe fatty degeneration and intracellular necrosis in the liver and pancreas but not in the heart. Biochemically, the content of triglycerides in the liver of mice treated with desulfated yessotoxin increased about 60 times, and phospholipids two-fold more than the control levels of those of mice treated with yessotoxin.

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.

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), (13)C- and (19)F-labelled covalent conjugates 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-C(2)-Erg 3 is similar to that of AmB in ergosterol-containing membranes, but significantly larger than that of AmB in nonsterol or cholesterol-containing membranes. 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-C(2)-(6-F)Cho 4, which are composed of uniformly (13)C-labelled AmB and 6-fluorinated 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 A. 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.

The synthesis of the CDEF ring fragment of yessotoxin, a marine ladder polyether, has been achieved. Union of three components, the C ring aldehyde, the F ring diol, and trimethylsilyl cyanide, was accomplished by treatment with Sc(OTf) 3 to afford the α-cyano ether both in stepwise and one-pot reactions. The DE ring system was constructed through a ring closing metathesis reaction and reductive etherification sequence.

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 Mn2+ 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.

Correction for ‘Stereoselective synthesis of the head group of archaeal phospholipid PGP-Me to investigate bacteriorhodopsin–lipid interactions’ by Jin Cui, et al., Org. Biomol. Chem., 2015, DOI: 10.1039/c5ob01252j.

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 (EC 5 0 , 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.

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-(13)C,(15)N]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.

Structure elucidation studies on natural products are reviewed emphasizing extremely small sample amounts. Previous studies on insect pheromones, periplanones, and bean-originating kairomones, glycinoeclepins, are described briefly. Recent examples are selected from marine natural products such as ciguatoxin, dolastatin-3, and aurisides. A more detailed description is given of a sperm-activating and attracting factor (SAAF), which may be the smallest sample amount used in the structure elucidation of novel non-peptidic natural products. SAAF was isolated from the eggs of the ascidian, Ciona intestinalis, and its structure was deduced with only approximately 4 microg (6 nmol) of sample. Based upon the proposed structure, two epimers were synthesized from chenodeoxycholic acid in 17 steps, leading to the identification of SAAF as a novel sterol sulfate.

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.