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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

yūki gōsei kagaku kyōkaishi/yuuki gousei kagaku kyoukaishi

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