Truncated derivatives of amphidinol 3 reveal the functional role of polyol chain in sterol-recognition and pore formation.

Here we examined the membrane binding and pore formation of amphidinol 3 (AM3) and its truncated synthetic derivatives. Importantly, both of the membrane affinity and pore formation activity were well correlated with the reported antifungal activity. Our data clearly demonstrated that the C1-C30 moiety of AM3 plays essential roles both in sterol recognition and stable pore formation. Based on the current findings, we updated the interacting model between AM3 and sterol, in which the moiety encompassing from C21 to C67 accommodates a sterol molecule with forming hydrogen bonds with the sterol hydroxy group and van der Waals contact between AM3 polyol and sterol skeleton. Although the conformation of the C1-C20 moiety of AM3 is hard to specify due to its flexibility, the region likely contributes to stabilization of pore structure.

Total Synthesis of Amphidinol 3: A General Strategy for Synthesizing Amphidinol Analogues and Structure-Activity Relationship Study.

Amphidinol 3 (AM3) is a potent antifungal produced by the dinoflagellate Amphidinium klebsii. It was difficult to determine the absolute configuration of AM3 by using the scarce natural product due to the presence of numerous stereogenic centers on the acyclic carbon chain. Since the absolute configuration was partially determined on the basis of insufficient evidence, the originally proposed structure has been revised three times. Although recent progress on structure determination by computational analysis is remarkable, total synthesis is still the most reliable way to confirm structures. The first total synthesis of AM3 was achieved via expeditious assembly of three components in five steps, confirming the revised structure of AM3 after more than 20 years since its first discovery. The established synthetic route would be a general strategy for synthesizing amphidinol congeners. An artificial and simplified analogue of AM3, which elicited antifungal activity comparable to that of AM3, was designed and synthesized. This is the first example of a biologically active artificial analogue possessing a shorter polyol moiety, providing insight on the antifungal mode-of-action.

Synthesis and Complete Structure Determination of a Sperm-Activating and -Attracting Factor Isolated from the Ascidian Ascidia sydneiensis.

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 25 S; thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved.

Synthesis and Stereochemical Revision of the C31-C67 Fragment of Amphidinol†3.

Amphidinol 3 (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.

Syntheses and Biological Activities of the LMNO, ent-LMNO, and NOPQR(S) Ring Systems of Maitotoxin.

Structure-activity relationship studies of maitotoxin (MTX), a marine natural product produced by an epiphytic dinoflagellate, were conducted using chemically synthesized model compounds corresponding to the partial structures of MTX. Both enantiomers of the LMNO ring system were synthesized via aldol reaction of the LM ring aldehyde and the NO ring ketone. These fragments were derived from a common cis-fused pyranopyran intermediate prepared through a sequence involving Nozaki-Hiyama-Kishi reaction, intramolecular oxa-Michael addition, and Pummerer rearrangement. The NOPQR(S) ring system, in which the original seven-membered S ring was substituted with a six-membered ring, was also synthesized through the coupling of the QR(S) ring alkyne and the NO ring aldehyde and the construction of the P ring via 1,4-reduction, dehydration, and hydroboration. The inhibitory activities of the synthetic specimens against MTX-induced Ca2+ influx were evaluated. The LMNO ring system and its enantiomer induced 36 and 18% inhibition, respectively, at 300 µM, whereas the NOPQR(S) ring system elicited no inhibitory activity.

Design and synthesis of benzoacridines as estrogenic and anti-estrogenic agents.

Estrogens play undisputedly important physiological roles, but lifetime exposure to estrogens has also been linked to the development of breast cancer. Moreover, imbalanced estrogen levels have been associated with various symptoms such as osteoporosis and menopausal disorders. For the improvement of such estrogen imbalances, estrogenic reagents with regulatory properties have shown promising potential. Herein, we report the construction of a 12-arylbenzoacridine library via a diversity-oriented strategy that furnished non-toxic estrogenic and anti-estrogenic agents. Derivatives with a hydroxy group at the molecular edge exhibit potent binding affinity to the estrogen receptor α (ERα) and ERß (IC50 < µM), while binding to the estrogen-related receptor γ (ERRγ), i.e., an orphan nuclear receptor on which estrogens often trigger unfavorable events, was not observed. These findings offer valuable insights into 12-arylbenzoacridines as a novel platform for the development of selective estrogen-receptor modulators (SERMs).

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.

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 (19)F-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 (13)C{(19)F} 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.

Axial hydrogen at C7 position and bumpy tetracyclic core markedly reduce sterol's affinity to amphotericin B in membrane.

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 (13)C{(19)F} 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.

Stereoselective synthesis of the C1-C29 part of amphidinol 3.

Stereoselective synthesis of the C1-C29 part of amphidinol 3 (AM3) was achieved. The C1-C20 part was assembled from three building blocks via regioselective cross metathesis to form the C4-C5 double bond and addition of an alkenyllithium and a lithium acetylide to two Weinreb amides followed by asymmetric reduction to form the C9-C10 and C14-C15 bonds, respectively. The C21-C29 part was synthesized via successive cross metathesis and oxa-Michael addition sequence to construct the 1,3-diol system at C25 and C27 and Brown asymmetric crotylation to introduce the stereogenic centers at C23 and C24. Coupling of the C1-C20 and C21-C29 parts was achieved by Julia-Kocienski olefination and regio- and stereoselective dihydroxylation of the C20-C21 double bond in the presence of the C4-C5 and C8-C9 double bonds to afford the C1-C29 part of AM3.

Synthesis and biological activity of the C'D'E'F' ring system of maitotoxin.

Stereoselective synthesis of the C'D'E'F' ring system of maitotoxin was achieved starting from the E' ring through successive formation of the D' and C' rings based on SmI2-mediated reductive cyclization. Construction of the F' ring was accomplished via Suzuki-Miyaura cross-coupling with a side chain fragment and Pd(II)-catalyzed cyclization of an allylic alcohol. The C'D'E'F' ring system inhibited maitotoxin-induced Ca(2+) influx in rat glioma C6 cells with an IC50 value of 59 µM.

Synthesis of the MN Ring of Caribbean Ciguatoxin C-CTX-1 via Desymmetrization by Acetal Formation.

The MN ring of Caribbean ciguatoxin C-CTX-1 was synthesized from a meso-syn-2,7-dimethyloxepane derivative corresponding to the M ring via desymmetrization by acetal formation with a camphor derivative, followed by construction of the N ring via the Horner-Wadsworth-Emmons reaction and acetal formation. The meso-syn-2,7-dimethyloxepane derivative was synthesized via photoinduced electrocyclization of a conjugated exo-diene under flow conditions, giving a cyclobutene derivative, followed by ring expansion via oxidative cleavage and diastereoselective reduction of a ß-hydroxy ketone.

Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane.

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.

Comprehensive molecular motion capture for sphingomyelin by site-specific deuterium labeling.

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.

Optical precursors in coupled-resonator-induced transparency.

We experimentally examined the propagation of temporally square modulated optical pulses through a coupled ring resonator. Sharp transient spikes appeared as the square pulses entered the system. The main signal gradually grew up through coupled-resonator-induced transparency (CRIT), with the time constant determined by a second resonator. Transient spikes were attributed to the higher and lower spectral components of the incident pulse, to which the resonators cannot respond; hence, they were interpreted as optical precursors. The experiments, therefore, demonstrated that precursors and the main signal can be observed separately, with amplitudes comparable to that of the incident step in CRIT.

Artificial ladder-shaped polyethers that inhibit maitotoxin-induced Ca2+ influx in rat glioma C6 cells.

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.

NMR-based conformational analysis of sphingomyelin in bicelles.

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.

Confirmation of the absolute configuration at C45 of amphidinol 3.

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.

Amphotericin B assembles into seven-molecule ion channels: An NMR and molecular dynamics study.

Amphotericin B, an antifungal drug with a long history of use, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol ergosterol. A stable assembly consisting of seven drug molecules was observed to form an ion conductive channel. The structure is somewhat similar to the upper half of the barrel-stave model proposed in the 1970s but substantially different in the number of molecules and in their arrangement. The present structure explains many previous findings, including structure-activity relationships of the drug, which will be useful for improving drug efficacy and reducing adverse effects.

Design and synthesis of sphingomyelin-cholesterol conjugates and their formation of ordered membranes.

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.

Conformations of spermine in adenosine triphosphate complex: the structural basis for weak bimolecular interactions of major cellular electrolytes.

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 ß- 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.