Showing papers by "Michio Murata published in 2018"

Synthesis and Stereochemical Revision of the C31–C67 Fragment of Amphidinol 3

Abstract: 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.

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

Abstract: 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.

Nanosized Phase Segregation of Sphingomyelin and Dihydrosphigomyelin in Unsaturated Phosphatidylcholine Binary Membranes without Cholesterol

Abstract: In this study, we applied fluorescence spectroscopy, differential scanning calorimetry (DSC), and 2H NMR to elucidate the properties of nanoscopic segregated domains in stearoylsphingomyelin (SSM)/dioleoylphosphatidylcholine (DOPC) and dihydrostearoylsphingomyelin (dhSSM)/DOPC binary membranes. The results obtained from fluorescence measurements suggest the existence of gel-like domains with high fluidity in both SSM and dhSSM macroscopic gel phases. The DSC thermograms showed that DOPC destabilizes SM-rich gel-like domains to a much lesser extent compared to the same amount of cholesterol. It was also found that a stable lateral segregation occurs without cholesterol, indicating that SSM itself undergoes homophilic interactions to form small gel-like domains. 2H NMR experiments disclosed differences in the temperature-dependent ordering of SSM/DOPC and dhSSM/DOPC bilayers; the dhSSM membrane showed less miscibility with the DOPC fluid phase, higher thermal stability, and tighter packing. In addition, the...

Recent Solid-State NMR Studies of Hydrated Lipid Membranes

Abstract: 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.

NMR studies on natural product-stereochemical determination and conformational analysis in solution and in membrane

Abstract: 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.

A Synthetic Approach to the Channel Complex Structure of Antibiotic in a Membrane: Backbone 19F-Labeled Amphotericin B for Solid-State NMR Analysis

Abstract: Over 40 years have passed since a well─ known hypothesis of a channel─ like amphotericin B (AmB) assembly with ergosterol (Erg) was proposed as the mode of action accounting for its selective fungal toxicity. However, no reliable or direct experimental evidence had been obtained until recently mainly because of signi cant dif culties in structural analysis of the self─ assembly of small molecules speci cally formed inside a membrane. In this study, we describe the accomplishments in the chemical synthesis of two AmB derivatives with uorine labeling in the molecular skeleton for applying solid─ state NMR techniques. The interatomic distance measurements using these chemically prepared probes have successfully shown the detailed AmB─ Erg bimolecular interaction in the channel structure for the rst time. The latest solid─ state NMR analysis backed by synthetic chemistry is expected to achieve a breakthrough in elucidating the long─ unsolved AmB channel architecture. Figure 1. (a) Chemical structure of amphotericin B, ergosterol, and cholesterol. (b) Conceptual illustration of AmB channels. Vol.76 No.11 2018 ( 61 ) 1197 acids. Especially for AmB, which has various functional groups, introducing an isotope label selectively into the desired position through both chemical modi cation and biosynthetic method is dif cult. Moreover, a single─ atom label should be site─ speci cally installed in the framework to obtain accurate structural information, and this often necessitates comparable efforts to those of total synthesis; 9 particularly, the total synthesis of AmB has been achieved only by Nicolaou group. 10 Nonetheless, as we reviewed recently, solid─ state NMR techniques seem to be the most appropriate way. 11 Figure 1 illustrates one of the possible models for an AmB channel complex, called a single─ length channel, where a single AmB─ Erg complex passes through the bilayer membrane. 12─ 14 Under these circumstances, we envisioned that overcoming the challenging synthesis of labeled AmB derivatives is the only way to make a breakthrough in the structural elucidation. Before starting this synthetic study for solid─ state NMR, we made several important ndings on the properties of a membrane─ bound channel assembly of AmB and Erg. For example, we prepared several covalent dimers of AmB and examined their membrane activity, 15─ 19 which revealed that AmB channels have orientation─ dependent recti cations for an ion ux through the channel. 19 Another synthetic approach to the ion channel function of AmB is the use of intramolecular bridged derivatives to restrict the rotational conformation of the sugar (mycosamine) moiety, indicating that the orientation and the possible hydrogen bond between 2’─ OH of the sugar and 3─ OH of Erg are important for AmB to exert its selective toxicity. 20,21 In this review, we rst discuss the design, synthesis, and biological evaluation of labeled AmB derivatives, with a focus on a uorine atom as a useful atomic label for solid─ state NMR measurements. Then, we will describe some representative examples of AmB─ Erg interaction analysis in the AmB channel complex by using synthesized F─ AmB derivatives with the labeled Erg. 2. Synthesis of Backbone Fluorine─ labeled AmB Derivatives Among NMR─ sensitive nuclei, uorine has signi cant advantages, such as its high gyromagnetic ratio and low background signals in nature, which enable a measurement of longer interatomic distances. 22 However, its high electronegativity often causes adverse effects on physicochemical properties and biological activities. 23 Particularly in the case of small molecules, the perturbation by uorine becomes more profound than that in relatively large molecules, such as peptides and polysaccharides. Therefore, we should design and synthesize a target compound by taking the following three points into consideration: 1) there should be no signi cant in uence on its bioactivity, 2) it should have a relevant position to the channel structure, and 3) facile introduction should be used. 2.1 Synthesis of 14─ 19 F─ AmB to Measure the C─ F Distance between the Hydrophilic Head Groups of AmB Although the substitution of a hydroxy group with a uorine atom seems to be better, given the effect on biological activity, modifying one of the multiple hydroxy groups in a site─ and stereo─ selective manner may be quite dif cult for AmB. In addition, uorine labeling with a different orientation, that is, not on the same side as polyhydroxy groups facing the inner pore, is preferable to obtain an intermolecular distance relevant to the channel structure. Along with the demand for backbone labeling, we designed a uorine─ labeled AmB derivative at the C14 position, a part of the polar THP ring (14─ F─ AmB, 2, Figure 2), as a synthetic target; as shown in Figure 11, the hydroxy groups largely face the opposite direction from the Erg molecule, whereas the 14─ position comes closer to Erg in the complex. In this position, the perturbation to the channel structure by the uorine atom was considered insigni cant because it was surrounded by polar hydroxy groups. Moreover, it has facile and selective accessibility based on the adjacent C13 hemiacetal structure via glycal formation and its electrophilic uorination, and it is also useful to obtain detailed structural information on the head group of AmB. Our synthetic strategy for 14─ F─ AmB is shown in scheme 1. After Fmoc protection of AmB, treatment with TMSOTf led to glycal formation and subsequent TMS deprotection with HF─ pyridine afforded 13,14─ anhydro derivative 3. Then, electrophilic uorination of the glycal moiety was performed using Select uor 24 to give the desired uorinated hemiacetal compound in a stereoselective manner, which was followed by the removal of the Fmoc group and HPLC puri cation to complete the synthesis of 14─ F─ AmB. The stereochemistry of uorinated position C14 was unambiguously determined to be S Figure 2. Structure of 14─ F─ AmB. Scheme 1. Reagents and conditions: (a) FmocOSu, DMF, pyridine, 25 °C, 18 h, quant; (b) TMSOTf, 2,6─ lutidine, CH 2Cl 2, 25 °C, 40 min; (c) HF─ pyridine, THF, 25 °C, 4 h, 50% (two steps); (d) Select uor, DMF─ H 2O (3:1), 25 °C, 1 h, 30%; (e) piperidine, DMSO─ MeOH (4:1), 25 °C, 1 h, 50% (isolation yield), Fmoc: 9─ Fluorenylmethyloxycarbonyl. ( 62 ) J. Synth. Org. Chem., Jpn. 1198 con guration by large vicinal (axial) coupling constants of H14 to H17 in the chair conformation. The biological evaluation demonstrated that 14─ F─ AmB showed hemolytic and antifungal activities comparable to those of AmB, which indicated the successful preparation of 14─ F─ AmB as an NMR probe for the structural analysis of AmB self─ assembly. 25 2.2 Synthesis of 32─ 19 F─ AmB to Measure the C─ F Distance between the Hydrophobic Sides (and tail parts) of AmB To elucidate the channel structure, particularly AmB─ Erg interaction more precisely, not only 14─ F─ AmB but also another F─ labeled─ AmB at the tail position became necessary. In other words, a labeled AmB at the hydrophobic part turned out to be very important for elucidating AmB─ Erg and AmB─ phospholipid interactions, including AmB─ AmB ones. Then, we designed a uorine─ labeled AmB derivative at the C32 position, an outer part of the heptaene moiety (32─ F─ AmB, 4, Figure 3), as the next synthetic target. Labeling at this position, the opposite side of C14 at a head group, to elucidate hydrophobic interactions in the outer part of a channel and around the tail part, which would be a versatile NMR probe. However, in addition to the concern of a possible perturbation to biological activity because of uorine substitution in the low polar ole n moiety ( uoroole n is regarded as a polar isostere of amide ), an expected considerable synthetic effort that is almost equal to total synthesis was the most signi cant problem to be solved because uorination to this position was never achieved by a simple modi cation of AmB, as in the case of 14─ F─ AmB. We then planned a practical and ef cient synthesis of 32─ F─ AmB by using a hybrid synthetic strategy with a combination of degradation of the natural product and chemical synthesis. 27,28 Namely, a polyol segment will be prepared with a cutout from commercially available AmB, which allowed us to skip multiple reaction steps for the construction of many chiral centers. On the other hand, a polyene segment was prepared by pure chemical synthesis, which ideally enabled us to introduce the labeling into an arbitrary position. The retrosynthesis of 32─ F─ AmB is shown in Scheme 2. The macrolactone backbone was to be synthesized via Stille coupling and following macrolactonization reaction of polyol C1─ C21 segment with a protected micosamine moiety (5) and a uorinated polyene C22─ C37 segment (6). The C1─ C21 segment (5) was to be prepared by oxidative removal of the C21─ C37 carbon chain from the fully protected AmB derivative. The C22─ C37 segment (6) would be synthesized from the known lactone 9 29 by a sequential carbon elongation using Horner─ Wadsworth─ Emmons (HWE) reaction. The preparation of the C1─ C21 segment commenced with the protection of natural AmB (Scheme 3). After AmB was Figure 3. Structure of 32─ F─ AmB. Scheme 2. Retrosynthesis of 32─ F─ AmB. MP: p ─ methoxyphenyl, SEM: 2─ (Trimethylsilyl)ethoxymethyl, TBS: t ─ Butyldimethylsilyl, EE: 1─ Ethoxyethyl. Scheme 3. Reagents and conditions: (a) Fmoc─ OSu, pyridine, DMF, 25 °C, overnight; (b) SEMCl, Na 2CO 3, DMF, 0 °C, 2 h, 77% (two steps); (c) p─ methoxybenzaldehyde dimethyl acetal, CSA, MeOH, 25 °C, 2 h, 96%; (d) TBSOTf, 2,6─ lutidine, CH 2Cl 2, -50 °C to 0 °C, 2 h, 73%; (e) O 3, -78 °C, 10 min, then PPh 3, 25 °C, 3 h, 59%; (f) CrCl 2, CHI 3, THF, 25 °C, 11 h, 64%; (g) LiOH, THF, H 2O, MeOH, 25 °C, 21.5 h; (h) Fmoc─ OSu, pyridine, DMF, 25 °C, 5 h, 35% (two steps). Vol.76 No.11 2018 ( 63 ) 1199 treated with FmocOSu, carboxylic acid was protected with a 2─ (trimethylsilyl)ethoxymethyl (SEM) group. In previ