The Structure of the Bimolecular Complex between Amphotericin B

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Structure of Bimolecular Complex between Amphotericin B and Ergosterol in Membrane is Stabilized by Face-toFace Van der Waals Interaction with their Rigid Cyclic Cores Yasuo Nakagawa, Yuichi Umegawa, Naohiro Matsushita, Tomoya Yamamoto, Hiroshi Tsuchikawa, Shinya Hanashima, Tohru Oishi, Nobuaki Matsumori, and Michio Murata Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00193 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 29, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Structure of Bimolecular Complex between Amphotericin B and Ergosterol in Membrane is Stabilized by Face-to-Face Van der Waals Interaction with their Rigid Cyclic Cores Yasuo Nakagawa, Yuichi Umegawa, Naohiro Matsushita, Tomoya Yamamoto, Hiroshi Tsuchikawa, Shinya Hanashima, Tohru Oishi,† Nobuaki Matsumori†,* and Michio Murata*

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.

Corresponding Author * Phone (+81)-6-6850-5774, Fax (+81)-6-6850-5774, e-mail [email protected] (M.M.), and Phone (+81)-9-2642-2582, e-mail [email protected] (N.M.).

This study was partly supported by JSPS KAKENHI Grant Number 25242073 and Grant Number 25750383, and also in part as ‘Lipid Active Structure Project (ERATO)’ from Japan Science and Technology Agency.

1 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

ABBREVIATIONS AFM, atomic force microscope; AmB, amphotericin B; Erg, ergosterol; CP, cross-polarization; CODEX, centerband-only detection of Exchange; DIBAL, diisobutylaluminium hydride; H-bond, hydrogen bond; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, High performance liquid chromatography; MAS, magic angle spinning; MCMM, Multi-Corner MultiMode; MNBA, 2-methyl-6-nitrobenzoic anhydride;

SEM, 2-(trimethylsilyl)ethoxymethyl;

TBAF,

tert-butyldimethylsilyl;

Tetra-n-butylammonium

fluoride;

TBS,

TMSE,

2-

(trimethylsilyl)ethyl; TNCG, Truncated Newton Conjugate Gradient; TPPM, Two Pulse Phase Modulation; OPLS, Optimized Potentials for Liquid Simulations; POPC, 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine; ppm, parts per million; PRCG, Polak-Ribiele conjugate gradient; REDOR, rotational-echo double-resonance; VDW, van der Waals.

Present Addresses †Department of Chemistry, Graduate School of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan


Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ABSTRACT

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 NMR, 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 group of AmB, the fully deprotected labeled-AmB compounds were obtained successfully. Then, these labeled AmBs were subjected to

13

C{19F} 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, whose ratio was 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 face-to-face, 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.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

In the past 50 years, amphotericin B (AmB, Figure 1A), a polyene macrolide produced by Streptomyces nodosus, has been used as a standard drug for the treatment of deep-seated systemic fungal infections. Generally, the antifungal activity and selective toxicity of AmB can be attributed to the formation of ion-permeable channels in the cell membrane in the presence of sterols; AmB exhibits a higher affinity to ergosterol (Erg)-containing fungal membranes than cholesterol (Cho)-containing mammalian membranes. On the other hand, the drug’s moderate selectivity for Erg over Cho causes severe side effects such as nephrotoxicity and liver damage.1,2 In the 1970s, De Kruijff et al. proposed a well-known “barrel-stave” model as the ionchannel complex comprising eight pairs of AmB and Erg.3 As evidenced by atomic force microscopy imaging,4 surface pressure measurements,5 and surface plasmon resonance,6 Erg efficiently stabilizes a membrane-bound form of AmB.7,8 Cohen et al. reported that the highconductance ion channels of AmB appear at a lower concentration in the Erg-containing membrane than in the Cho-containing membrane.9 Our studies also showed that Cho decreases the ion-flux activity of membrane-bound AmB, while increasing the membrane binding of the drug from the aqueous phase.6,10 Recently, Burke et al. proposed a ‘sterol sponge model’ in which they suggested that the sequestering of Erg from cell membranes mainly contributes to the antifungal activity of the drug, while the ion-channel formation of AmB was regarded as a secondary action that further increased the drug potency.11 In either method, the drug’s selective toxicity to fungal cells can be attributed to its higher affinity to Erg. To improve the therapeutic index of the drug, it is important to elucidate the structure of the AmB–Erg pair responsible for its higher affinity. Therefore, the structure elucidation of the molecular assemblies of an AmB/Erg complex in membranes has long been considered as the greatest challenge in the mode-of-action study of the drug.


Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Solid-sate NMR techniques have long been applied to antimicrobial peptides in lipid bilayers and the membrane-binding motifs of proteins, and led to important findings, most of which cannot be obtained by other methods.12–16 On the other hand, the membrane-bound structures of nonpeptidic molecules, particularly natural products, have hardly been investigated by NMRbased methods. This limited application of solid-state NMR can be attributed to several reasons, e.g., difficulties in synthesizing isotope-labeled compounds, lack of fundamental knowledge on their membrane behavior, and the formation of a mixture with various oligomeric states. Thus, in addition to the pharmaceutical significance, the high-resolution structure of the AmB–sterol complex could lead to a breakthrough in the mechanism-of-action studies on diverse natural products with membrane activities.17–20 Recent studies on the antimicrobial activity of synthetic AmB derivatives by Carreira,21 the structure–activity relationship analysis of conformation-restricted derivatives by our group,22 and the molecular dynamics simulation by Baginski23 indicated that the hydrogen bonding between the mycosamine moiety of AmB and the 3-OH group of sterol plays an essential role in bimolecular interactions. Burke11d has recently reported a similar parallel interaction between the AmB of Cho based on a synthetic deoxyl derivative of AmB at the 2′-OH group of mycosamine that resulted in the development of an AmB-based drug with improved pharmaceutical properties. As shown by these previous studies, the parallel orientation of AmB–Erg (Figure 1B) has been established as their interaction mode. In the structural studies on the membrane-bound assembly of AmB/sterol, we focused on solid-state NMR,24 along with affinity measurements and computational analysis.23,25 To further investigate the AmB–Erg interactions by solid-state NMR, we have previously carried out 13

C{19F} rotational-echo double-resonance (REDOR) experiments using 14-F-AmB (Figure


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1A)26 and

13

Page 6 of 37

C-labeled Erg obtained biosynthetically.24c Although the measurements were partly

hampered by the low intensity and heavy overlapping of presence of multiple

13

C NMR signals because of the

13

C-labeled sites in Erg, these results indicate that both “parallel” and

“antiparallel” pairs are present in the AmB–Erg complex formed in membranes (Figure 1B). To further confirm this unexpected orientation, site-specifically labeled 26,27-13C2-Erg (Figure 1A) was prepared to measure the 13C{19F}REDOR with 14-F-AmB; the REDOR results indicate that the antiparallel orientation comprises 30% of the total AmB–Erg pairing based.24c Moreover, the distance between the 19F atom of 14-F-AmB and the 13C26/13C27 atoms of Erg in “antiparallel” orientation was also successfully estimated. Our next objective was to measure further intermolecular distances for both the orientations to obtain a clear image of the AmB–Erg bimolecular complex. Interatomic distance for the head-to-head contact is essential information since the parallel orientation is assumed to facilitate the interaction between 3-OH of Erg and the headgroup of AmB.21-23 In addition,

19

F-labeling at the bottom half of an AmB molecule is

necessary to obtain the distance constraint for the tail-to-tail contact in the parallel complex (Figure 1). The preparation of

19

F-labeled AmB bearing a free carboxylic acid at the C41

position, which is reported to be crucial for the Erg selectivity of AmB in phospholipid layers,27,28 is thought to be much more difficult than that of AmB methyl etster.29 The Erg/Choselective activity of AmB derivatives in artificial membrane systems does not always correlate to the in vivo pharmacological properties; AmB methyl ester and its derivatives are known to show comparable antifungal and antiviral activities with less nephrotoxicity,30 but its Erg/Cho selectivity is usually lower than AmB upon measuring ion-flux activity with artificial liposomes.27,28,31


Page 7 of 37

In this study, we synthesized a new site-specifically

19

F-labeled AmB derivative, 32-F-AmB

(Figure 1A), by combining the fragments obtained from chemical synthesis and the degradation of the natural product via cross-coupling and macrolactonization reactions. The labeled compounds, mixed with 4-13C-Erg or 26,27-13C2-Erg (Figure 1A), was subjected to intermolecular 13C{19F}REDOR measurements. Based on the distance information obtained, the structure of bimolecular AmB–Erg complexes was proposed for the first time on the basis of interatomic distance data.

OH

OH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. (A) Chemical structures of AmB 1, 14-F-AmB 2, 26,27-13C2-Erg 3, 4-13C-Erg 4, and newly synthesized labeled-AmB, 32-F-AmB 5. (B) Illustrations for bimolecular parallel and antiparallel orientations between AmB (yellow) and Erg (blue). The parallel orientation involves “head-to-head” and “tail-to-tail” contacts of AmB/Erg and the antiparallel orientation involves “head-to-tail” contact of AmB/Erg.

MATERIAL AND METHODS


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

Materials: AmB was purchased from Nacalai Tesque (Kyoto, Japan). Erg was purchased from Tokyo Kasei (Tokyo, Japan), and palmitoyl oleoyl phosphatidylcholine (POPC) was purchased from Avanti Polar Lipid Inc. (Alabaster, AL). All other chemicals were purchased from standard venders. 26,27-13C2-Erg 3 and 4-13C-Erg were synthesized from Erg as reported previously.24c,32 The synthetic details of 32-F-AmB are shown in Supporting Information.

Preparation of multilamellar vesicle for solid-state NMR: 19F-labeled-AmB 2 or 5 (1.7 mg, 1.8 µmol; in the case of dilution experiment, half the amount of

19

F-labeled-AmB was replaced by

natural AmB), 13C-labeled-Erg 3 or 4 (720 µg, 1.8 µmol), and POPC (12.3 mg, 16.1 µmol) were dissolved in a mixture of CHCl3/MeOH, and the solvent was evaporated to afford a thin film. After standing in vacuo for 8 h, the membrane was hydrated with 14.7 µL of 10 mM HEPES buffer (pH 7.0) and 500 µL of H2O and dispersed by vortexing and sonication. Then, the lipid suspension was freeze-thawed five times to produce large vesicles. The suspension was lyophilized, rehydrated with D2O (14.7 µL), and packed into a glass tube. The glass tube was sealed using epoxy glue and inserted into an MAS rotor of φ 5 mm. For the solid-state NMR measurement, the liposome samples were also prepared in the same manner, while the labeled positions of AmB and Erg were different.

Solid-state NMR measurements: The 13C{19F}REDOR spectra were recorded using a CMX300 (Agilent Technologies, Santa Clara, CA, USA) spectrometer at 75 MHz 13C resonance frequency with a MAS frequency of 5000 ± 2 Hz. The temperature-controlling air was maintained at 30 ± 1 °C; the temperature in a sample rotor was increased to 38 °C.24c The spectral width was 30 kHz. Typically, the π/2 pulse widths of 1H were 5.0 and 3.1 µs for 26,27-13C2-Erg and 4-13C-Erg,


Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

respectively; the π-pulse widths for

13

C and

19

F were 6.7 and 9.3 µs. The contact times for the

cross-polarization transfer were set at 1.0 ms. The REDOR spectra were acquired with a recycle delay of 2 s under TPPM 1H-decoupling33 with a field strength of 50.0 or 80.6 kHz, and xy-8 phase cycling34 was used for 19F. Typically, the number of scans of each experiment was 20,000.

Molecular modeling of AmB–Erg interaction: The conformational search was carried out with the Macromodel software version 9.9 installed on a Windows 7 operating system. The initial atomic coordinates of AmB were obtained from the crystal data of N-iodoacetyl AmB.35 The initial atomic coordinates of Erg were provided by a molecular mechanics simulation. The conformational search and energy minimization of Erg were performed using the OPLS 200536 (Macromodel 9.9) as described in our previous paper.32,37 One of the most stable conformer whose side chain was fully extended was used as the initial conformation of Erg. During the simulation, the carbon backbone of AmB was frozen, while the hydroxyl groups, amino group, and carboxyl group were rotated to form hydrogen bonds. The side chain of Erg was restricted as the initial conformation with a width of ±30°. Other sterol atoms were calculated without any restriction. For the calculation of parallel interactions, the initial geometry was set as parallel. The distance constraints between the hydrogen and 13C with a width of 0.2, which is the typical difference in C–H and C–F bond distances, were used instead of those between 19F and 13C; the interatomic distance between the hydrogen atoms of AmB and 13C atoms of Erg were restricted by H14/C4 = 6.4±0.2 Å and H32/C26,27 = 5.2, 6.8±0.2 Å, respectively. The force constant of the restriction was at 200 kJ/mol Å. The calculation was carried out with two inverted constraints of F32/C26,27 = 5.2, 6.8 Å or 6.8, 5.2 Å because the distance from C26 and C27 was interchangeable. Considering the conformers from both the calculations, AmB–Erg interaction


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

was discussed. These two types of calculations were also carried out for the antiparallel manner with restrictions by F32/C4 = 6.0 Å and F32/C26,27 = 5.5, 7.0 Å. The conformational space was sampled by a Monte Carlo multiple minimum method (MCMM),38 and the position and torsion angle of Erg were optimized in vacuum. The OPLS 2005 force field implemented in the Macromodel program was used for the conformational searches in 20,000 steps. Energy minimization with the same constraint as the conformational search was carried out using the Polak-Ribiere conjugate gradient (PRCG) method39 with 28,000 maximum iterations.

RESULTS AND DISCUSSION Synthesis of 19F-labeled AmB. We have previously reported the synthesis of 28-F and 25-13C AmB methyl ester by a combination of chemical synthesis (C22–C37 segment) and degradation of natural AmB (C1–C21 segment).29 In this study, we needed to prepare a free carboxylic acid form of AmB because it is well known that the carboxylic acid form of C41 is important for the sterol-selectivity and ion-channel formation.28 We envisaged that labeled AmB 5 can be synthesized via Stille coupling and macrolactonization by a similar synthetic strategy (Scheme 1) as that reported in our previous study,29 and C1–C21 segment 7 (Scheme 2) can be prepared via the degradation of natural AmB. Although in our previous report,29 the carboxylic acid group of compound 7 was protected as a methyl ester, its selective deprotection appeared to be difficult due to the concomitant ring-opening of macrolactone.31 In the new synthetic scheme, the carboxylic acid group was protected as 2-(trimethylsilyl)ethoxymethyl (SEM) ester that can be selectively removed by fluorides such as TBAF and HF.40 For the synthesis of C1–C21 7 shown in scheme 2, the previous report29b was followed except protecting the carboxylic acid group as an SEM ester instead of a methyl ester. The degradation


Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

of natural AmB via (i) the protection of the amino group with an Fmoc group and the carboxylic acid group as an SEM ester and (ii) the selective protection of the 1,3-diols (3,5- and 9,11positions) as p-methoxybenzylidene (MP) acetals and the remaining hydroxy groups as TBS ethers afforded fully protected AmB 6. (iii) The exhaustive ozonolysis of the heptaene moiety and the subsequent Takai olefination41,42 of the resulting aldehyde and (iv) selective hydrolysis in the presence of an SEM ester and the reprotection of the amino group afforded C1–C21 segment 7. Although the cleavage of the SEM group was partially observed during the TBS protection and hydrolysis, C1–C21 segment 7 was obtained in acceptable yields.

Scheme 1. Retrosynthesis of 32-F-labeled AmB


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

Scheme 2. Reagents and conditions: (a) Fmoc-OSu, pyridine, DMF, rt, overnight; (b) SEMCl, Na2CO3, DMF, 0 °C, 2 h, 77% (two steps); (c) p-methoxy benzaldehyde dimethyl acetal, CSA, MeOH, rt, 2 h, 96%; (d) TBSOTf, 2,6-lutidine, CH2Cl2, −50 °C to 0 °C, 2 h, 73%; (e) O3, −78 °C, 10 min, then PPh3, rt, 3 h, 59%; (f) CrCl2, CHI3, THF, rt, overnight, 64%; (g) LiOH, THF, H2O, MeOH, rt, 21.5 h; (h) Fmoc-OSu, pyridine, DMF, rt, 5 h, 35% (two steps).

The polyol fragment was successfully synthesized, and thus the stage was set to start the synthesis of the polyene part followed by the reconstruction of an AmB skeleton and deprotection (Scheme 3). The fluorinated polyene part, C22–C37 segment 16, was synthesized from aldehyde 8.29a The Horner–Wadsworth–Emmons (HWE) reaction with fluorophosphonate 9 proceeded stereoselectively afforded fluoroolefin 10 with E geometry. Olefin 10 was converted to triene ester 13a in three steps, comprising reduction with DIBAL, oxidation with Dess–Martin periodinane, and HWE reaction with unsaturated phosphonate 12.43 In the next step, E-olefin 13a (E,E,E) was isomerized to the desired Z-olefin 13b (Z,E,E) by irradiation with a tungsten lamp in the presence of PhSeSePh.29a Refluxing in toluene accelerated the isomerization reaction, 12 ACS Paragon Plus Environment

Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

completing the conversion within 20 h while concomitantly eliminating an ethoxyethyl group. After the reprotection with the same group, the ester was further converted to trienal 14 by DIBAL

reduction

and

subsequent

Dess–Martin

oxidation.

The

HWE

reaction

of

stannylphosphonate 1544 with trienal 14 afforded the fluorinated polyene segment 16. Then, following the previous procedure,29 the Stille coupling between 7 and 16, macrolactonization, and deprotection were conducted. As expected, the SEM group tolerated the TBS deprotection conditions with HF-pyridine. Finally, 32-F-AmB was well purified by HPLC, thus completing the synthesis. We also confirmed that the potency of antifungal and hemolytic activities and the sterol-dependent permeabilization of bilayer membranes by 32-F-AmB were comparable with those of natural AmB (Table S1 and Figure S8), clearly indicating that the introduction of a fluorine atom at the C32 of AmB did not influence its recognition mechanism of Erg and Cho in membranes.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

Scheme 3. Reagent and conditions: (a) 9, n-BuLi, THF, −78 °C, 97%; (b) DIBAL, THF, −78 °C; (c) Dess–Martin periodinane, CH2Cl2, rt, 2 h, 93% (two steps); (d) 12, LHMDS, THF, −78 °C, 1 h, 92%; (e) PhSeSePh, hν, toluene, 20 h, reflux; (f) ethyl vinyl ether, PPTS, CH2Cl2, rt, 1.5 h, 77% (two steps); (g) DIBAL, THF, −78 °C; (h) Dess–Martin periodinane, CH2Cl2, rt, 2 h, 82% (two steps) (i) 15, LHMDS, 0 °C, 40 min, 63%; (j) C1–C21 segment 7, Pd2(dba)3·CHCl3, AsPsPh3, i-Pr2NEt, THF, rt, 4 h; (k) PPTS, p-methoxy benzaldehyde dimethyl acetal, CH2Cl2, rt, 3 h; (l) MNBA, DMAP, CH2Cl2, rt, 4 h, 28% (three steps); (m) HF-pyridine, MeOH, 50 °C, 40 h; (n) piperidine, CH2Cl2, rt, 5 h; (o) HCl, MeOH, 0 °C, 20 min, then HCl, H2O, t-BuOH, 0 °C, 5 h, 17 % (three steps).

Thus, we established an efficient synthetic route for fully deprotected labeled AmB with a free carboxylic acid group by protecting the carboxylic acid group as an SEM ester in this study, affording 32-F-AmB by protecting the carboxylic acid group as an SEM ester. Recently, Burke synthesized 35-deoxy-AmB by a similar method, combining chemical synthesis and the degradation of natural AmB.11b


Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Distance measurements of

13

C and

19

F-labeling sites between AmB and Erg in parallel

interaction. In the next step, the 13C{19F}REDOR45 spectra of hydrated membrane preparations containing two different pairs of AmB and Erg, 14-F-AmB/4-13C-Erg and 32-F-AmB/26,2713

C2-Erg, were recorded to obtain the distance constraints for both the terminal parts in the

parallel orientation. Figures 2A/C show the full-echo (S0) and difference spectra (∆S) of the REDOR experiments, where F-AmB and

13

C-Erg were mixed in a 1:1 ratio in hydrated POPC

bilayers. C26/27 of Erg produced two peaks at δ 21.5 and 23.0 ppm (Figure 2C), which were assigned to a free form and an AmB-bound form. The signals of C4 from free (black arrows) and bound Erg (red arrows) were observed at δ 42.3 and 39.7 ppm.24c,32 The large intensity difference between the bound and unbound forms of the C4 peak (Figure 2A) can be attributed to the very short T2 of C4 in the AmB-bound form; the T2 relaxation of a methylene carbon is known to be greatly accelerated in a reduced molecular mobility. As expected, significant REDOR dephasings were observed for the broad peak only due to the formation of AmB–Erg complex. These dephasing effects clearly indicate that only the 13C atoms in an AmB−Erg complex were significantly dephased by pulses applied at the

19

F frequency band. In other words, the parallel

interaction between AmB and Erg certainly occurs in a hydrated POPC membrane at room temperature. Figures 2B and 2D show the plots (filled circle) of experimental REDOR dephasing data (∆S/S0) for the C4 and C26/C27 signals, respectively. Notably, the dephasing effect was saturated at ∼70% for both the 13C-labeled Ergs, indicating that ∼70% of Erg binds to AmB in a parallel manner.24c The REDOR plots were fitted to the theoretical curves to estimate the distance between F32/C4 and F32/C26,27.46 It is well known that the dephasing of the 13C signal in 13C{19F}REDOR is influenced by the number of spins and molecular motion.47 Therefore, to estimate the interatomic distances more correctly, further REDOR experiments were carried out


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

with a different F-AmB/13C-Erg ratio or at a low temperature as described in the following section.

Effect of the other fluorine atom and molecular motion on REDOR dephasing. First, to investigate the number of fluorine atoms that cause the REDOR dephasing of

13

C signals, the

concentration of 14-F-AmB was diluted with nonlabeled AmB (1) at the AmB/14-F-AmB/4-13CErg ratio of 0.5/0.5/1 for 13C{19F}REDOR measurements. The dephasing values of the 13C signal of 4-13C-Erg (Figure 2B, open circles) for this diluted system were half of those of the undiluted systems. These results indicate that the only nearest

13

C-Erg/19F-AmB pair virtually influenced

the REDOR dephasing (the details are provided in Supporting Information), allowing to calculate the interatomic distance based on a simple 13C–19F two-spin system. Next, to evaluate the molecular motion including the exchange of AmB-sterol pairing during the

13

C{19F}REDOR experiments, similar experiments were carried out at −30 °C using 14-F-

AmB/4-13C-Erg/POPC = 1:1:9. It is known that a rapid exchange in the pairing of irradiated/observed spins such as

19

F and

13

C in our experiments attenuates their dipole–dipole

interaction easily. Therefore, if the AmB–Erg pairing frequently exchanges at 38 °C, the dephasing effect of

13

C signal should be significantly reduced compared to that obtained at

−30 °C. The obtained REDOR dephasing effects at a low temperature (Figure S4) were slightly enhanced, however, mostly similar to those obtained under physiological conditions, indicating that the exchange of AmB–Erg pairing rarely occurs during the REDOR measurement even at 38 °C (further discussion on molecular motion is given in Supporting Information). A similar long lifetime of the interaction was also observed for the AmB–POPC and Erg–POPC complexes in our previous REDOR experiments.24a,c These results strongly indicate that AmB, Erg, and


Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

POPC form a very stable complex in lipid bilayers. From the theoretical curve fitting24c,46 (Figures 2B and 2D), the time-averaged distance between C4 and F14 was estimated to be 6.4±0.1 Å. For the signal of C26/27, the dephasing effect was the sum of the two pairs of spins, C26/F14 and C27/F14. Thus, the F-32/C26 and F-32/C27 distances were estimated to be 5.2±0.1 and 6.8±0.1 Å, respectively; the values are interchangeable because the C26 and C27 signals heavily overlapped. It is important to estimate the relative fractions of unbound and bound AmB and Erg. We measured the MAS spectra of 14-F-AmB as shown in Figure S5, which demonstrated the two components with different mobility. The sharp component with faster mobility may be due to axially symmetrically moving F-AmB as in the case of membrane lipids while the powder pattern-like component may be due to F-AmB in the assembly that moves very slowly and gives rise to the REDOR dephasing effect.

The

13

C spectra of 26,27-13C2-Erg under dipolar

decoupling conditions (DD-MAS, Figure S6) allowed us to estimate the ratio of bound and unbound Erg to be 1:3.3. Together with the F-AmB results, the molar fraction of AmB bound to Erg was estimated to be around 25-30% (See Supporting Information for details).


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6.4 Å

Page 18 of 37

5.2 Å

6.8 Å

Figure 2.

13

C{19F}REDOR spectra of 4-13C-Erg/14-F-AmB (A) and 26,27-13C2-Erg/32-F-AmB

(C) at a 1:1:9 ratio of F-AmB/13C-Erg/POPC. S0(black line), S(red line), and ∆S correspond to a full-echo spectrum, REDOR-dephased spectrum, and different spectrum, respectively. The asterisks indicate the signal from POPC. Both the spectra were obtained after a

19

F-dephasing

period of 32 rotor cycles (12.8 ms) at 38 °C. The black and red arrows indicate the signals due to the free and bound Erg, respectively. *Signals due to POPC. (B) Experimental 13C{19F}REDOR dephasing values for the C4 signal of 14-F-AmB/4-13C-Erg = 1:1 (●) and nonlabeled AmB/14F-AmB/4-13C-Erg = 0.5:0.5:1 (○). The simulation curves for

13

C−19F distances of 6.4 Å. (D)

Experimental 13C{19F}REDOR dephasing values (●) for the C26/27 signal of 32-F-AmB/26,2713

C2-Erg = 1:1 and simulation curves (solid lines) for the 13C−19F distances of 5.2 and 6.8 Å. The


Page 19 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

dashed line is the simple average of the two solid lines for 5.2 and 6.8 Å: the 13C–19F two-spin systems were assumed for each pair, while the REDOR curves for

13

C/13C–19F three-spin

systems were similar to those of the two-spin systems (data not shown).

Distance between AmB and Erg in antiparallel interaction. For the antiparallel alignment, the intermolecular distances between the head-group of AmB and the side chain of Erg have been reported as shown in Figure 3C/D.24c However, to obtain a picture of the AmB–Erg complex, another distance constraint is necessary. Thus,

13

C{19F} REDOR experiments were

carried out using 4-13C-Erg and 32-F-AmB to obtain the distance between the tail part of AmB and the Erg core was obtained. Figure 3A shows the full-echo (S0) and difference spectrum (∆S), where 4-13C-Erg and 32-F AmB were mixed in a 1:1 ratio in hydrated POPC bilayers. As expected, a significant REDOR dephasing was observed for the broad peak of bound Erg only at δ 39.7 ppm. Figure 3B shows the experimental REDOR dephasing values (∆S/S0) for the C4 peak at different dephasing times. The dephasing effect reached a plateau at ∼30%, which corresponded well to the 7:3 ratio of parallel and antiparallel orientation for AmB–Erg pairs. From the curve fitting of REDOR dephasing, the distance between the C4 of Erg and the F32 of F-AmB was estimated to be 6.0±0.2 Å.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

5.5 Å

6.0 Å

7.0 Å

Figure 3. 13C{19F}REDOR spectra of 4-13C-Erg/32-F-AmB/POPC (A) and 26,27-13C2-Erg/14-FAmB (C) at a 1:1:9 ratio of F-AmB/13C-Erg/POPC. S0(black line), S(red line), and ∆S correspond to a full-echo spectrum, REDOR-dephased spectrum, and different spectrum, respectively. Both the data were obtained after a

19

F-dephasing period of 32 rotor cycles (12.8 ms) at 38 °C. The

black and red arrows indicate the signals from the free and bound Erg, respectively. (B) Experimental 13C{19F}REDOR dephasing values (●) for C4 signal and the simulation curves for 13

C−19F distances of 6.0 Å. *Signals due to POPC. (D) Experimental 13C{19F}REDOR dephasing

values (●) for C26/27 signal of 14-F-AmB/26,27–13C2–Erg = 1:1 and simulation curves (solid lines) for the

13

C−19F distances of 5.5 and 7.0 Å. The simulation curve (dashed line) derived

from two different distances 5.5 and 7.0 Å (1:1). §Spectrum C and REDOR curve D are the same


Page 21 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

as reported previously; 24c the spectra were obtained with a small modification in the window function.

Molecular modeling of AmB–Erg bimolecular interaction. These 13C{19F}REDOR results, along with those obtained previously, provided the three distances for each parallel and antiparallel manners of AmB–Erg interaction. The obtained distances are listed in Table 1. Using these intermolecular distance constraints, the topological analysis of AmB–Erg bimolecular alignment was carried out based on a conformational search. As the initial structures, the X-ray crystal structure of AmB35 and energy-minimized structure of Erg32,37 were used. During the conformational search, the carbon backbone of AmB was frozen, while the hydroxyl groups, amino group, and carboxyl group were rotated to facilitate hydrogen-bond formation. The mycosamine moiety was also restricted to a “closed form” as reported by Neumann et al.23b The side chain of Erg was almost restricted to an extended form to enhance the VDW interaction with AmB.32,37 Under the interatomic distance constrains obtained from the REDOR experiments (Table 1), MD simulations were carried out using the conformational search program on Macromodel 9.9.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table1. Intermolecular distances between F-AmB and

Page 22 of 37

13

C-Erg calculated from the

13

C{19F}

REDOR experiments. Atomic position Distance (Å) a F14 (AmB) - C4 (Erg) 6.4±0.1 F32 (AmB) - C26 or C27(Erg) 5.2±0.1 or 6.8±0.1 Anti-paralle F32 (AmB) - C4 (Erg)24c 6.0±0.2 F14 (AmB) - C26 or C27(Erg) 5.5±0.2 or 7.0±0.2 a Errors were estimated by the S/N ratio of the S0 and ∆S spectra.

Interaction manner Parallel

After the conformational search, 10 and 33 conformers were obtained within 10 kJ mol−1 from the ground minimum in parallel and antiparallel orientations, respectively. These conformers were categorized into four groups according to their relative alignments. In the first group (Figures 4A and 4E), the hydrophobic plane of AmB (opposite side of mycosamine moiety) faces the flat plane of Erg. In the second group (Figures 4B and 4F), the hydrophobic plane of AmB faces the angular methyl groups of Erg. In the third group (Figures 4C and 4G), the angle between the macrolide plane of AmB and the steroid plane of Erg is perpendicular. In the fourth group (Figure 4D), Erg is closer to the mycosamine moiety. The antiparallel alignment of this group was only found in a higher energy level than 10 kJ mol−1 (Figure 4H).


Page 23 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4. Side views of parallel and antiparallel alignments generated by conformational search. The obtained structures were categorized into four groups (A, B, C, and D for parallel alignment and E, F, G, and H for antiparallel alignment) and superimposed. Within 10 kJ mol−1 from the


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

ground minimum, in the parallel alignment, two, four, one, and three conformers were obtained for A, B, C, and D groups, respectively, and in the antiparallel alignment, 6, 16, and 11 conformers were obtained for E, F, and G groups, respectively. Alignment H was observed as the minor geometry with a higher potential energy. I: - and β-faces of Erg. In the bottom panels, the structures of the most stable complexes for parallel (a) and antiparallel (e) orientations in the side and top views that were picked up from A and E in the top panel, respectively, are shown as space-filling models. *C7 position of Erg (see Supporting Information for details).

Most of the conformers obtained for both the parallel and antiparallel alignments can be categorized into four groups A/E, B/F, C/G, and D/H shown in Figure 4, in which the Erg is located beside the hydrophobic plane of the macrolactone ring of AmB, but interacts with AmB using different faces. These alignments do not match the traditional barrel–stave models, in which an Erg molecule interacts with the mycosamine moiety of AmB.21,22,23b,48,49 We recently reported that a flat steroid ring system of Erg facilitates a close face-to-face VDW contact with the macrolide ring of AmB, while a non-planar tetracyclic core and an axial hydrogen atom at C7 on the -side of the sterol core significantly reduce the affinity to AmB.32 These results strongly indicate that the -face of an Erg alicycle (Figure 4I) interacts with AmB. Moreover, the REDOR experiments indicate that the parallel and antiparallel AmB–Erg orientations occur at a 7:3 ratio in assemblies. Therefore, probably both the orientations of AmB–Erg are mainly stabilized by a common interaction mode such as face-to-face VDW, while more polar interactions such as hydrogen bonding may not be the main factor. In fungus membranes, where Erg occurs in both the outer and inner leaflets of bilayers, the parallel and antiparallel interaction of AmB and Erg


Page 25 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

may facilitate AmB to extract Erg from either leaflet of cell membranes.24c Evaluating the complex structures in Figure 4 with regard to the close face-to-face interaction between AmB and Erg, we concluded that complex structures A and E are most plausible conformations for their parallel and antiparallel interactions, respectively. The structure of the stable complex a (Figure 4) implies that the face-to-face interaction could account for the drug’s selectivity of Erg over Cho since the 7-axial hydrogen of Cho could hamper the close contact between the rigid macrocycle of AmB and the alicyclic core of sterol.32 Another interesting point of the structure a is that the 2’-OH of mycosamine directs away from the 3-OH of Erg although the orientation of a mycosamine moiety, for which we used the X-ray data,35 was thought to be favorable for interaction with Erg.22 This result implies that, since Erg is supposed to be sandwiched between two molecules of AmB in the barrel stave model, the sterol hydroxyl group may possibly interact with another molecule of AmB residing in the other side of Erg, for which we could not obtain distance data. In addition to the intermolecular distance, the precise conformation of the mycosamine moiety with respect to the aglycon of AmB has to be determined. Regarding the interaction of antiparallel Erg with the AmB molecule in the opposite side, no experimental data were obtained from the present study; in the previous studies,23a,24a the parallel AmB-AmB orientation was suggested to be dominant in ergosterol-containing membrane, which implies that Erg interacts with the two molecules of AmB on both sides in the antiparallel manner. To finally answer this question, we will have to wait until the whole structure of an AmB/Erg assembly is solved. It is interesting to infer the relevance of the present results to the sterol sponge model,11 in which AmB molecules are supposed to interact tightly with Erg. As described in Figure S1-S3, the nearest

13

C/19F pair that mostly causes the REDOR dephasing are due to the AmB/Erg


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

complexes in a VDW contact, hence implying that these bimolecular interactions could occur even in the phospholipid-free aggregates like the sterol sponge. In this study, we proposed the structure of an AmB–Erg biomolecular complex for the first time based on the interatomic distances obtained from the experimental REDOR data. Nevertheless, some pieces of information are still missing to precisely elucidate the mechanism of the sterol-specific recognition by the drug. For example, one Erg molecule possibly interacts with more than two AmB molecules to reinforce the channel assembly, and the involvement of phospholipids may be another important factor.24d,50 We have previously detected the interaction between AmB and phospholipid using

13

C{31P} REDOR experiments.24c,24d In these studies,

REDOR dephasing effects were observed between the side chain of AmB-bound ergosterol and the phosphate group of membrane lipid, and between the C1-branching groups at both of the terminal portions of AmB and the phosphate. The findings imply that membrane phospholipids firmly interact with the AmB-ergosterol complex and play an essential role in stabilizing the channel assembly. To address these problems, the configurations of AmB–AmB and AmBphospholipid complexes as well as the orientation of the mycosamine moiety are currently investigated.

CONCLUSION We synthesized

19

F-labeled AmB with a free carboxyl group by using an SEM ester as the

SEM protecting group. The 32-F-AmB is expected to be a powerful tool to investigate not only AmB–Erg interactions, but also AmB–AmB interactions and oligomeric number.51,52 Moreover, this method can be used to synthesize other AmB derivatives labeled with various NMRsensitive nuclei such as

19

F,

13

C, and 2H at different positions; these labeled AmBs would


Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

provide more detailed information. The 13C{19F}REDOR measurements using 14-F-AmB, 32-FAmB, 4-13C-Erg, and 26,27-13C2-Erg clearly indicate that the parallel and antiparallel interactions concurrently occur in lipid bilayers at a ratio of 7:3. The three intermolecular distances for each parallel and antiparallel interactions were successfully estimated. The conformational search using the REDOR-derived distance constraints showed that AmB and Erg in the parallel and antiparallel alignments interact with each other in a similar manner. The flat face of Erg and the mycosamine unit of the macrolide of AmB closely contact each other, thus facilitating a face-to-face VDW contact in either parallel or antiparallel orientation. These interactions may account for the coexistence of the two AmB-Erg complexes at 7:3 ratio. These findings on the mechanism of specific AmB-Erg interaction may provide essential information for enhancing the drug’s utility by reducing its severe side effects. In addition, their close faceto-face VDW interaction may lead to a better understating of the sterol recognition mechanism by other natural compounds.

ACKNOWLEDGMENT Y. N. was a JSPS fellow supported by Japan Society for the Promotion of Science.

SUPPORTING INFORMATION AVAILABLE Other solid-state NMR experiments and analysis including dilution experiments and low temperature measurements of REDOR, conformation search for AmB-cholesterol complex, the synthesis and biological activity and sterol selectivity of 32-F-AmB. This material is available free of charge via the Internet at http://pubs.acs.org.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

REFERENCES (1)

Hartsel, S., and Bolard, J. (1996) Amphotericin B: new life for an old drug. Trends Pharmacol. Sci. 17, 445–449.

(2)

Lemke, A., Kiderlen, A. F., and Kayser, O. (2005) Amphotericin B. Appl. Microbiol. Biotechnol. 68, 151–162.

(3)

De Kruijff, B., and Demel, R. A. (1974) Polyene antibiotic-sterol interactions in membranes of acholeplasma laidlawii cells and lecithin liposomes. III. molecular structure of the polyene antibiotic- cholesterol complexes. Biochim. Biophys. Acta 339, 57–70.

(4)

Milhaud, J., Ponsinet, V., Takashi, M., and Michels, B. (2002) Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol. Biochim. Biophys. Acta 1558, 95–108.

(5)

Barwicz, J., and Tancrède, P. (1997) The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers. Chem. Phys. Lipids 85, 145–155.

(6)

Mouri, R., Konoki, K., Matsumori, N., Oishi, T., and Murata, M. (2008) Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance. Biochemistry 47, 7807–7815.

(7)

Kamiński, D. M. (2014). Recent progress in the study of the interactions of amphotericin B with cholesterol and ergosterol in lipid environments. Eur. Biophys. J., 43, 453-467.

(8)

Kotler-Brajtburg, J., Price, H., Medff, G., Schlessinger, D., and Kobayashi, G. S. (1974) Molecular basis for the selective toxicity of amphotericin B for yeast and filipin for animal cells. Antimicrobial Agents and Chemotherapy 5, 377–382.


Page 29 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(9)

Cohen, B. E. (1992) A sequential mechanism for the formation of aqueous channels by amphotericin B in liposomes. The effect of sterols and phospholipid composition. Biochim. Biophys. Acta 1108, 49–58.

(10) Matsuoka, S., and Murata, M. (2002). Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B. Biochim. Biophys. Acta 1564, 429-434. (11) (a) Palacios, D. S., Anderson, T. M., and Burke, M. D. (2007) A post-PKS oxidation of the amphotericin B skeleton predicted to be critical for channel formation is not required for potent antifungal activity. J. Am. Chem. Soc. 129, 13804–13805. (b) Gray, K. C., Palacios, D. S., Dailey, I., Endo, M. M., Uno, B. E., Wilcock, B. C., and Burke, M. D. (2012) Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. U. S. A. 109, 2234–2239. (c) Palacios, D. S., Dailey, I., Siebert, D. M., Wilcock, B. C., and Burke, M. D. (2011) Synthesis-enabled functional group deletions reveal key underpinnings of amphotericin B ion channel and antifungal activities. Proc. Natl. Acad. Sci. U. S. A. 108, 6733–6738. (d) Wilcock, B. C., Endo, M. M., Uno, B. E., and Burke, M. D. (2013) C2’-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells. J. Am. Chem. Soc. 135, 8488–8491. (e) Anderson, T. M., Clay, M. C., Cioffi, A. G., Diaz, K. A., Hisao, G. S., Tuttle, M. D., Nieuwkoop, A. J., Comellas, G., Maryum, N., Wang, S., Uno, B. E., Wildeman, E. L., Gonen, T., Rienstra, C. M., and Burke, M. D. (2014) Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10, 400–406. (f) Davis, S. A., Vincent, B. M., Endo, M. M., Whitesell, L., Marchillo, K., Andes, D. R., Lindquist, S., and Burke, M. D. (2015). Nontoxic antimicrobials that evade drug resistance. Nat. Chem. Biol. 11, 481-487.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

(12) Das, N., Dai, J., Hung, I., Rajagopalan, M., Zhou, H., and Cross, T. (2014). Structure of CrgA, a cell division structural and regulatory protein from mycobacterium tuberculosis, in lipid bilayers. Proc. Natl. Acad. Sci. U. S. A. 112, E119–E126. (13) Wadhwani, P., Strandberg, E., Van den Berg, J., Mink, C., Bürck, J., Ciriello, R. A., and Ulrich, A. S. (2014). Dynamical structure of the short multifunctional peptide BP100 in membranes. Biochim. Biophys. Acta 1838, 940-949. (14) Lee, D.-K., Bhunia, A., Kotler, S. A., and Ramamoorthy, A. (2015). Detergent-type membrane fragmentation by MSI-78, MSI-367, MSI-594, and MSI-843 antimicrobial peptides and inhibition by cholesterol: a solid-state nuclear magnetic resonance study. Biochemistry 54, 1897-1907. (15) Higman, V. A., Varga, K., Aslimovska, L., Judge, P. J., Sperling, L. J., Rienstra, C. M., and Watts, A. (2011). The conformation of bacteriorhodopsin loops in purple membranes resolved by solid-state MAS NMR spectroscopy. Angew. Chem. Int. Ed. 50, 8432-8435. (16) Liao, S. Y., Yang, Y., Tietze, D., and Hong, M. (2015). The influenza M2 cytoplasmic tail changes the proton-exchange equilibria and the backbone conformation of the transmembrane histidine residue to facilitate proton conduction. J. Am. Chem. Soc 137, 6067-6077. (17) Bolard, J. (1986) How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim. Biophys. Acta 864, 257–304. (18) Sugiyama, R., Nishimura, S., Matsumori, N., Tsunematsu, Y., Hattori, A., and Kakeya, H. (2014) Structure and biological activity of 8-deoxyheronamide C from a marine-derived Streptomyces sp.: heronamides target saturated hydrocarbon chains in lipid membranes. J. Am. Chem. Soc. 136, 5209–5212.


Page 31 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(19) (a) Houdai, T., Matsuoka, S., Matsumori, N., and Murata, M. (2004) Membranepermeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate. Biochim. Biophys. Acta 1667, 91–100. (b) Houdai, T., Matsuoka, S., Morsy, N., Matsumori, N., Satake, M., and Murata, M. (2005). Hairpin conformation of amphidinols possibly accounting for potent membrane permeabilizing activities. Tetrahedron 61, 2795-2802. (c) Morsy, N., Houdai, T., Matsuoka, S., Matsumori, N., Adachi, S., Oishi, T., Murata, M., Iwashita, T., and Fujita, T. (2006) Structures of new amphidinols with truncated polyhydroxyl chain and their membrane-permeabilizing activities. Bioorg. Med. Chem. 14, 6548–6554. (d) Espiritu, R. A., Matsumori, N., Tsuda, M., and Murata, M. (2014) Direct and stereospecific interaction of amphidinol 3 with sterol in lipid bilayers. Biochemistry 53, 3287–3293. (20) (a) Nishimura, S., Arita, Y., Honda, M., Iwamoto, K., Matsuyama, A., Shirai, A., Kawasaki, H., Kakeya, H., Kobayashi, T., Matsunaga, S., and Yoshida, M. (2010) Marine antifungal theonellamides target 3beta-hydroxysterol to activate Rho1 signaling. Nat. Chem. Biol. 6, 519–526. (b) Espiritu, R. A., Matsumori, N., Murata, M., Nishimura, S., Kakeya, H., Matsunaga, S., and Yoshida, M. (2013) Interaction between the marine sponge cyclic peptide theonellamide A and sterols in lipid bilayers as viewed by surface plasmon resonance and solid-state 2H nuclear magnetic resonance. Biochemistry 52, 2410–2418. (21) Croatt, M., and Carreira, E. (2011) Probing the role of the mycosamine C2′-OH on the activity of amphotericin B. Org. Lett. 13, 1390–1393. (22) Matsumori, N., Sawada, Y., and Murata, M. (2005) Mycosamine orientation of amphotericin B controlling interaction with ergosterol: sterol-dependent activity of


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 37

conformation-restricted derivatives with an amino-carbonyl bridge. J. Am. Chem. Soc. 127, 10667–10675. (23) (a) Neumann, A., Baginski, M., Winczewski, S., and Czub, J. (2013) The effect of sterols on amphotericin B self-aggregation in a lipid bilayer as revealed by free energy simulations. Biophys. J. 104, 1485–1494. (b) Neumann, A., Baginski, M., and Czub, J. (2010) How do sterols determine the antifungal activity of amphotericin B? Free energy of binding between the drug and its membrane targets. J. Am. Chem. Soc. 132, 18266–18272. (c) Neumann, A., Czub, J., and Baginski, M. (2009) On the possibility of the amphotericin Bsterol complex formation in cholesterol- and ergosterol-containing lipid bilayers: a molecular dynamics study. J. Phys. Chem. B 113, 15875–15885. (d) Baginski, M., and Czub, J. (2006) Modulation of amphotericin B membrane interaction by cholesterol and ergosterol – a molecular dynamics study. J. Phys. Chem. B 110, 16743–16753. (24) (a) Umegawa, Y., Matsumori, N., Oishi, T., and Murata, M. (2008) Ergosterol increases the intermolecular distance of amphotericin B in the membrane-bound assembly as evidenced by solid-state NMR. Biochemistry 47, 13463–13469. (b) Kasai, Y., Matsumori, N., Umegawa, Y., Matsuoka, S., Ueno, H., Ikeuchi, H., Oishi, T., and Murata, M. (2008) Self-assembled amphotericin B is probably surrounded by ergosterol: bimolecular interactions as evidenced by solid-state NMR and CD spectra. Chem. - Eur. J. 14, 1178– 1185. (c) Umegawa, Y., Nakagawa, Y., Tahara, K., Tsuchikawa, H., Matsumori, N., Oishi, T., and Murata, M. (2012) Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane. Biochemistry 51, 83–89. (d) Matsuoka, S., Ikeuchi, H., Matsumori, N., and Murata, M. (2005). Dominant formation of a single-length channel by amphotericin B in dimyristoylphosphatidylcholine membrane evidenced by


Page 33 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

13

C−

31

P rotational echo double resonance. Biochemistry 44, 704-710. (e) Matsumori, N.,

Tahara, K., Yamamoto, H., Morooka, A., Doi, M., Oishi, T., and Murata, M. (2009). Direct interaction between amphotericin B and ergosterol in lipid bilayers as revealed by 2H NMR Spectroscopy. J. Am. Chem. Soc 131, 11855-11860. (25) Baran, M., Borowski, E., and Mazerski, J. (2009) Molecular modeling of amphotericin Bergosterol primary complex in water II. Biophys. Chem. 141, 162–168. (26) Matsumori, N., Umegawa, Y., Oishi, T., and Murata, M. (2005) Bioactive fluorinated derivative of amphotericin B. Bioorg. Med. Chem. Lett. 15, 3565–3567. (27) Readio, J., and Bittman, R. (1982) Equilibrium binding of amphotericin B and its methyl ester and borate complex to sterols. Biochim. Biophys. Acta 685, 219–224. (28) (a) Hervé, M., Debouzy, J. C., Borowski, E., Cybulska, B., and Gary-bobo, C. M. (1989) The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A

31

P-NMR study. Biochim. Biophys. Acta 980, 261–

272. (b) Mazerski, J., Bolard, J., and Borowski, E. (1995) Effect of the modifications of ionizable groups of amphotericin B on its ability to form complexes with sterols in hydroalcoholic media. Biochim. Biophys. Acta 1236, 170–176. (29) (a) Tsuchikawa, H., Matsushita, N., Matsumori, N., Murata, M., and Oishi, T. (2006) Synthesis of 28-19F-amphotericin B methyl ester. Tetrahedron Lett. 47, 6187–6191. (b) Matsushita, N., Matsuo, Y., Tsuchikawa, H., Matsumori, N., Murata, M., and Oishi, T. (2009) Synthesis of 25-13C-amphotericin B methyl ester: a molecular probe for solid-state NMR measurements. Chem. Lett. 38, 114–115.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 37

(30) Waheed, A. A., Ablan, S. D., Mankowski, M. K., Cummins, J. E., Ptak, R. G., Schaffner, C. P., and Freed, E. O. (2006) Inhibition of HIV-1 Replication by Amphotericin B Methyl Ester: selection for resistant variants. J. Biol. Chem. 281, 28699–28711. (31) Szpilman, A. M., Cereghetti, D. M., Manthorpe, J. M., Wurtz, N. R., and Carreira, E. M. (2009). Synthesis and biophysical studies on 35-deoxy amphotericin B methyl ester. Chem. Eur. J. 15, 7117-7128. (32) Nakagawa, Y.,

Umegawa, Y.,

Nonomura, K.,

Matsushita, N.,

Takano, T.,

Tsuchikawa, Hanashima, S., Oishi, T., and Murata, M. (2015). Axial hydrogen at C7 position and bumpy tetracyclic core markedly reduce sterol’s affinity to amphotericin B in membrane. Biochemistry 54, 303–312. (33) Bennett, A. E., Rienstra, C. M., Auger, M., Lakshmi, K. V., and Griffin, R. G. (1995) Heteronuclear decoupling in rotating solids. J. Chem. Phys. 103, 6951–6958. (34) Gullion, T., Baker, D., and Conradi, M. (1990) New, compensated carr-purcell sequences. J. Magn. Reson. 89, 479–484. (35) Jarzembska, K. N., Kamin, D., Hoser, A. A., Malin, M., Senczyna, B., Wozniak, K., and Gagos, M. (2012) Controlled crystallization, structure, and molecular properties of iodoacetylamphotericin B. Cryst. Growth Des. 12, 2336–2345. (36) Kaminski, G., and Friesner, R. (2001) Evaluation and reparametrization of the OPLS-AA force field for proteins via comparison with accurate quantum chemical calculations on peptides. J. Phys. Chem. B 2, 6474–6487. (37) Nakagawa, Y., Umegawa, Y., Takano, T., Tsuchikawa, H., Matsumori, N., and Murata, M. (2014) Effect of sterol side chain on ion channel formation by amphotericin B in lipid bilayers. Biochemistry 53, 3088–3094.


Page 35 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(38) Chang, G., Guida, W., and Still, W. (1989) An internal-coordinate Monte Carlo method for searching conformational space. J. Am. Chem. Soc. 111, 4379–4386. (39) Polak, E. (1971) Computational methods in optimization; Academic Press: New York (40) Wuts, P.; Greene, T. (2007) Greene’s protective groups in organic synthesis 4 th ed., pp. 565–566. Wiley-Iterscience, New Jersey (41) Rogers, B., Selsted, M., and Rychnovsky, S. (1997) Synthesis and biological evaluation of non-polyene analogs of amphotericin B. Bioorg. Med. Chem. Lett. 7, 3177–3182. (42) Takai, K., Nitta, K., and Utimoto, K. (1986) Simple and Selective Method for RCHO (E)RCH=CHX Conversion by Means of a CHX3-CrCI2 System. J. Am. Chem. Soc. 108, 7408–7410. (43) Bennacer, B., Trubuil, D., Rivalle, C., and Grierson, D. S. (2003) The synthesis of two furan-based analogues of the α′,β′-epoxy ketone proteasome inhibitor eponemycin. European J. Org. Chem. 2003, 4561–4568. (44) Paquette, L. a., Pissarnitski, D., and Barriault, L. (1998) A modular enantioselective approach to construction of the macrolactone core of polycavernoside A. J. Org. Chem. 63, 7389–7398. (45) (a) Gullion, T., and Schaefer, J. (1989) Detection of weak heteronuclear dipolar coupling by rotational-echo double-resonance nuclear magnetic resonance. Adv. Magn. Reson. 13, 57–83. (42) Gullion, T., and Schaefer, J. (1989) Rotational-echo double-resonance NMR. J. Magn. Reson. 81, 196–200 (46) Mueller, K. (1995) Analytic solutions for the time evolution of dipolar-dephasing NMR signals. J. Magn. Reson. Ser. A 113, 81–93.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 37

(47) Matsuoka, S., and Inoue, M. (2009). Application of REDOR NMR in natural product chemistry. Chem. Commun. 5664-5675. (48) Silberstein, A. (1998) Membrane biology conformational analysis of amphotericin B – cholesterol channel complex. J. Membr. Biol. 126, 117–126. (49) Czub, J., Neumann, A., Borowski, E., and Baginski, M. (2009) Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives - A molecular dynamics study. Biophys. Chem. 141, 105–116. (50) (a) Matsuoka, S., and Murata, M. (2003). Membrane permeabilizing activity of amphotericin B is affected by chain length of phosphatidylcholine added as minor constituent. Biochim. Biophys. Acta 1617, 109-115. (b) Takano, T., Konoki, K., Matsumori, N., and Murata, M. (2009). Amphotericin B-induced ion flux is markedly attenuated in phosphatidylglycerol membrane as evidenced by a newly devised fluorometric method. Bioorg. Med. Chem., 17, 6301-6304. (51) DeAzevedo, E., and Hu, W. (1999) Centerband-only detection of exchange: efficient analysis of dynamics in solids by NMR. J. Am. Chem. Soc. 121, 8411–8412. (52) Luo, W., and Hong, M. (2006) Determination of the oligomeric number and intermolecular distances of membrane protein assemblies by anisotropic 1H-driven spin diffusion NMR spectroscopy. J. Am. Chem. Soc. 128, 7242–7251.


Page 37 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

For Table of Contents Use Only Structure of Bimolecular Complex between Amphotericin B and Ergosterol in Membrane is Stabilized by Face-to-Face Van der Waals Interaction with their Rigid Cyclic Cores Yasuo Nakagawa, Yuichi Umegawa, Naohiro Matsushita, Tomoya Yamamoto, Hiroshi Tsuchikawa, Shinya Hanashima, Tohru Oishi,† Nobuaki Matsumori†,* and Michio Murata*


The Structure of the Bimolecular Complex between Amphotericin B

Recommend Documents