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Thailandins A and B, New Polyene Macrolactone Compounds Isolated from Actinokineospora bangkokensis Strain 44EHW, Possessing Antifungal Activity against Anthracnose Fungi and Pathogenic Yeasts T

Bungonsiri Intra, Anja Greule, Andreas Bechthold, Jirayut Euanorasetr, Thomas Paululat, and Watanalai Panbangred J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2016 Downloaded from http://pubs.acs.org on June 7, 2016

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Journal of Agricultural and Food Chemistry

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1

Thailandins A and B, New Polyene Macrolactone Compounds Isolated

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from

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Antifungal Activity against Anthracnose Fungi and Pathogenic Yeasts

Actinokineospora

bangkokensis

Strain

44EHWT,

Possessing

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Bungonsiri Intra,†,‡ Anja Greule,# Andreas Bechthold,# Jirayut Euanorasetr,†,‡ Thomas

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Paululat,§ and Watanalai Panbangred *,†,‡

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†

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Thailand

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400,

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‡

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Biotechnology, Bangkok 10400, Thailand

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#

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Freiburg, Stefan-Meier-Str. 19, 79104 Freiburg, Germany

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§

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Reichwein-Str. 2, 57068 Siegen, Germany

Mahidol University and Osaka Collaborative Research Center on Bioscience and

Institute for Pharmaceutical Biology and Biotechnology, Albert-Ludwigs University of

Department of Chemistry-Biology, Organic Chemistry II, University of Siegen, Adolf-

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*Corresponding author (Tel: +66-20-15927; Fax: +66-20-15926; E-mail:

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[email protected]),

19 20 21 22

ACS Paragon Plus Environment


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ABSTRACT: Two new polyene macrolactone antibiotics, thailandins A, 1, and B, 2, were

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isolated from the fermentation broth of rhizosphere soil-associated Actinokineospora

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bangkokensis strain 44EHWT. The new compounds from this strain were purified using

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semi-preparative HPLC and Sephadex LH-20 gel filtration while following an antifungal

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activity guided fractionation.

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techniques including UV, HR-ESI-MS, and NMR. These compounds demonstrated broad

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spectrum antifungal activity against fungi causing anthracnose disease (Colletotrichum

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gloeosporioides DoA d0762, Colletotrichum gloeosporiodes DoA c1060 and Colletotrichum

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capsici DoA c1511) as well as pathogenic yeasts (Candida albicans MT 2013/1, Candida

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parasilopsis DKMU 434 and Cryptococcus neoformans MT 2013/2) with minimum

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inhibitory concentrations ranging between 16-32 µg/mL. This is the first report of polyene

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antibiotics produced by Actinokineospora species as bioactive compounds against

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anthracnose fungi and pathogenic yeast strains.

Their structures were elucidated through spectroscopic

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KEYWORDS: Actinokineospora, Actinokineospora bangkokensis, Rhizospheric soil,

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Polyene, Anthracnose

39 40 41 42 43 44 45 46


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INTRODUCTION

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The history of drug discovery reveals the potent ability of actinomycetes, Streptomyces as

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well as non-Streptomyces species, to produce a wide variety of antibiotics and other classes

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of biologically active secondary metabolites.1 However, finding new metabolites is greatly

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limited due to the fact that many terrestrial streptomycetes isolated from different

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environments produce compounds which have been previously isolated. Therefore, the focus

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on industrial screening for new bioactive compounds has moved to explore genera of non-

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Streptomyces, which are novel species.2-3 Novel and diverse bioactive secondary metabolites,

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such as complicated large macrocyclic compounds, are the most frequently isolated

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molecules in this new group of actinomycetes species.4

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The known secondary microbial metabolites from novel non-Streptomyces species possess

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many biological activities, including antimicrobial properties. Antifungal antibiotics are a

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significant group of drugs and have an important role in the control of fungal infection.

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However, the number of antifungal drugs is quite limited compared to the numerous classes

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of antibacterial agents. Antifungal drugs have wide application in the treatment of human

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fungal infections, agriculture, and veterinary medicine. There are five major classes of

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antifungal compounds: (i) polyene antibiotics, (ii) allylamines and thiocarbamates, (iii) azole

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derivatives, (iv) morpholines, and (v) nucleoside analogs.5 In the late 1950s, the first member

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of polyene macrolide antifungal antibiotics from Streptomyces species was discovered.6

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Polyene antifungal compounds such as amphotericin B were the standard of therapy for

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clinical fungal infection for several decades.

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Many fungal strains are important pathogens in agriculture and can cause several diseases

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in plants including anthracnose. Anthracnose disease is caused by several Colletotrichum

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spp. and is generally characterized by sunken necrotic tissue where orange conidial masses

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are produced.7 The disease results in severe losses of both pre- and post-harvest fruits in



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developing countries, particularly in Thailand, with a reduction in fruit yield of up to 80%.8

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Two significant causes of anthracnose disease in Thailand are the pathogens C. capsici and C.

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gloeosporioides.9 Plant pathogens in agriculture are generally controlled through the use of

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chemicals. However, the indiscriminate use of chemicals frequently leads to the evolution of

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resistant pathogens, environmental pollution, as well as adverse effects on useful soil insects

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and microorganisms. To overcome these problems, alternative control methods, including

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the use of biological microorganisms, have been attempted over the last 25-30 years.10-12

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Biocontrol of fungal plant diseases has been achieved using various antibiotics from

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actinomycetes, particularly Streptomyces species. Examples of such metabolites include

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macrolides, benzoquinones, aminoglycosides, polyenes and nucleosides.13-16 Most biological

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controls are directed toward wound pathogens and involve the use of antagonist bacterial

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strains that produce antibiotics.17

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Only few studies on secondary metabolite production from the member of the genus

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Actinokineospora have been reported.

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activity against Trypanosoma brucei brucei and actinosporins C–D with anti-oxidant activity

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were isolated from Actinokineospora spheciospongia EG49T.18-19 However, there are no

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previous reports on polyene antibiotics with antifungal activity from the genus

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Actinokineospora.

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rhizospheric soil collected in Bangkok, Thailand.20 This strain was found to be a novel

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species

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disease in plants. Here we describe the isolation, purification and structural elucidation of

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new polyene compounds produced by this newly isolated strain. In addition to activity

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against anthracnose disease, these compounds also displayed inhibitory activity against

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clinical yeast pathogens suggesting possible utility for these antifungal agents in both

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agricultural and medical settings.

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Recently, actinosporins A–B with anti-parasitic

We have previously isolated A. bangkokensis strain 44EHWT from

and displayed potent activity against fungal pathogens that cause anthracnose


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MATERIALS AND METHODS General procedure. All reagents used in this study were HPLC grade and analytical

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grade solvents.

Size exclusion chromatography was performed using Sephadex LH-20

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purchased from Sigma–Aldrich, Germany. LC-MS analysis of the samples was performed

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with an XCT 6330 LC/MSD Ultra Trap HPLC-ESI-MS system (Agilent, Waldbronn,

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Germany). Nuclear magnetic resonance spectra (NMR) spectra were measured on a VNMR-

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S 600 MHz spectrometer (Varian, Palo Alto, CA) equipped with 3mm triple resonance

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inverse and 3mm dual broadband probes. Spectra were recorded in 150 µL DMSO-d6.

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Solvent signals were used as an internal standard (DMSO-d6: δH 2.50, δC 39.5 ppm) and all

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spectra are recorded at 35 °C. Pulse sequences were taken from the Varian pulse sequence

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library.

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calibration based on sodium formate cluster.

HR-ESI-MS was performed on a Bruker Compact Mass Spectrometer, with

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Microorganisms and culture conditions. A. bangkokenesis strain 44EHWT (type strain

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44EHWT = BCC 53155T = NBRC 108932T) was incubated at 28 oC and maintained on

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International Streptomyces Project (ISP) media 2 and 4 (Difco, Sparks, MD). Cells grown on

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slants were subcultured to new media every 2 months. For long term preservation, spore

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suspension and cell culture broth in 20% glycerol were kept at -80 oC. Bacterial strains

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(Bacillus subtilis ATCC 6051 and Escherichia coli ATCC 25922) were incubated at 37 oC

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and kept in 15% glycerol at -80 oC for long term storage.

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Colletotrichum gloeosporioides DoA d0762, C. gloeosporioides DoA c1060 and C.

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capsici DoA c1511 were purchased from the Department of Agriculture, Ministry of

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Agriculture and Co-operatives, Thailand. Saccharomyces cerevisiae IFO 10217 was kindly

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provided by Dr. Chuenchit Boonchird, Department of Biotechnology, Faculty of Science,

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Mahidol University. The two clinical isolated yeast pathogens, Candida albicans MT 2013/1



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and Cryptococcus neoformans MT 2013/2 were kindly provided by Dr. Srisurang

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Tantimavanich, Faculty of Medical Technology, Mahidol University, Thailand. Candida

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parapsilosis DMKU 434 was obtained from Prof. Savitree Limthong, Faculty of Science,

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Kasetsart University. The fungi were cultivated and maintained on potato dextrose agar

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(PDA) (Merck, Darmstadt, Germany) at 30 oC and 37 oC, respectively.

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Phylogenetic analysis. PCR amplification and sequencing of the 16S rRNA genes were

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carried out as previously described21 with an ABI PRISM 3130 Genetic Analyzer (Applied

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Biosystems) according to the manufacturer’s instructions. The nearly full-length 16S rRNA

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gene sequence from the targeted strains were aligned with the corresponding sequences of all

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type strains in each genus using MUSCLE.22 Phylogenetic analyses were constructed with

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MEGA version 6.023 using tree-making algorithms, neighbour-joining24, maximum-

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parsimony25 and minimum-evolution26 methods. The trees topologies were evaluated by

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bootstrap analysis with 1000 re-samplings.27 The values of sequence similarities with closet

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strains were carried out using the EzTaxon server.28

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Comparison of the inhibitory activity of strain 44EHWT cultured in different media.

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To investigate a suitable medium for the production of active secondary metabolites, strain

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44EHWT was cultured in 100 mL of four different media. Medium utilized were HA (0.4%

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yeast extract, 1% malt extract and 0.4% glucose)29, NL5 (0.1% NaCl, 0.1% KH2PO4, 0.05%

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MgSO4.7H2O, 2.5% glycerol, 0.584% L-glutamine and 0.2% trace elements)30, MS (2% soya

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flour and 2% D-mannitol)31 and SG (2% glucose, 1% soya flour, 0.2% CaCO3, 0.1% CoCl2

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and 0.2% L-valine).32-33 Strain 44EHWT was pre-cultured in TSB medium (Difco, Sparks,

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MD) for 2 d at 28 oC and then further cultivated in the four media at 28 oC for 10 d. Crude

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extract samples were prepared separately from supernatants and pellets.

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obtained from strain 44EHWT cultivation were adjusted to pH 4 and pH 7, followed by two

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extractions with an equal volume of ethyl acetate. The organic phase was evaporated to


Supernatants

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dryness using rotary evaporation. Secondary metabolites from the pellet were separately

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extracted twice with an equal volume of acetone and followed by extraction with 1:1 volume

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of ethyl acetate:H2O. The organic phase was then evaporated to dryness. Subsequently,

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HPLC analysis was performed on an Agilent HP1200 system equipped with a photodiode

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array detector (200-600 nm) to identify samples from extracts and fractions with the desired

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activity as well as to evaluate the purity of the isolated compounds. HPLC analysis was

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performed on a Capcell Pak C18 (250 mm x 4.6 mm i.d., 5 µm) (Shiseido, Tokyo, Japan)

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using a gradient beginning with 5:95 acetonitrile:H2O to 95% acetonitrile in 36 min. Semi-

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preparative HPLC with UV detection was used for the isolation of pure compounds. Samples

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were collected separately in round-bottom flasks.

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Antimicrobial activities of crude extracts, fractions, and pure compounds were analyzed

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using the disk diffusion susceptibility test.

The indicator strains are as follows: Gram

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positive bacteria (B. subtilis ATCC 6051); Gram negative bacteria (E. coli ATCC 25922);

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fungi (C. gloeosporioides DOA d0762, C. gloeosporioides DOA c1060 and C. capsici DOA

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c1511); and yeast (C. albicans MT 2013/1, C. neoformans MT 2013/2, C. parapsilosis

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DMKU 434 and S. cerevisiae IFO 10217). Twenty microliters of crude extracts were loaded

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onto sterile blank filter disks (6 mm in diameter) and air-dried at room temperature. Disks

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were then transferred to Mueller-Hinton agar (MH) and Potato Dextrose agar (PDA)

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previously swabbed with indicator bacteria and fungi, respectively. The zone of growth

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inhibition was measured after incubation at 30 oC for 3-5 d for fungi and 37 oC overnight for

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bacteria.

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Fermentation, extraction, isolation and identification of antifungal polyene

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compounds. The fermentation broth from A. bangkokensis grown in HA medium (12 L) at

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28 oC for 10 d was extracted as described previously. Extracts from pellets and supernatants

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were combined and concentrated in vacuo. The crude extract (1.7 g) was then subjected to



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semi-preparative HPLC (HP 1260, Agilent Technologies). The HPLC column (250 mm x 10

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mm i.d., 5 µm, Capcell Pak C18 (Shiseido, Tokyo, Japan)) was eluted with H2O (A) and

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acetonitrile (B) using the following stepwise gradient: 30% B (3 min), 55-95% B (15 min),

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30% B (5 min). The flow rate was 2.5 mL/min. Peaks 1, 2, and 3 were collected at retention

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times of 12.1 min, 14.7 min, and 16.3 min, respectively. The fractions were lyophilized to

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dryness, dissolved with methanol, and then further purified using a Sephadex LH-20 (GE

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Healthcare, Uppsala, Sweden) column (22 cm x 2.5 cm diam.). Elution was performed using

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methanol and 1.5 mL was collected for each fraction. The target fractions were pooled and

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concentrated.

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performed using 1D and 2D NMR spectra together with mass spectrometry analysis.

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After obtaining the purified active compounds, structure elucidation was

Thailandin A, 1: 11.4 mg; yellow amorphous solid; HRESIMS m/z 755.4204 [M+H]+ (calcd for C39H63O14, 755.4212); 1H NMR and 13C NMR (see Table 1). Thailandin B, 2: 10.1 mg; yellow amorphous solid; HRESIMS m/z 609.3645 [M+H]+ (calcd for C33H53O10, 609.3633); 1H NMR and 13C NMR (see Table 1).

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Minimum inhibitory concentration (MIC) evaluation. The minimal inhibitory

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concentrations (MICs) of compounds 1 and 2 were determined using the broth microdilution

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method in 96-well microplates.

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Antimicrobial Susceptibility Testing (EUCAST) definitive document EDef 7.234 and 9.2.35

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For susceptibility tests, 100 µL of conidial suspensions at 2-5 x 106 conidia/mL for plant

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pathogenic fungi C. gloeosporioides DOA d0762, C. gloeosporioides DOA c1060 and C.

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capsici DOA c1511 were prepared. Samples of yeasts C. albicans and C. neoformans at 1-5

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x 106 CFU/mL were prepared based on the McFarland standard turbidity assay. Samples

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were further diluted 10-fold to obtain final working inoculums of 2-5 x 105 conidia/mL and

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1-5 x 105 CFU/mL for anthracnose fungi and yeasts, respectively. Compounds dissolved in

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DMSO (Merck, Hohenbrunn, Germany) were prepared using two-fold serial dilutions in

Procedures followed the European Committee on


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RPMI-1640 broth (Sigma-Aldrich, St. Louis, MO) to obtain concentrations ranging from 256

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µg/mL to 0.5 µg/mL. The conidial suspension of anthracnose fungi or yeast cultures (100

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µL) and compounds (100 µL) were added to individual wells of microplates.

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incubation at 30 oC for 3 d for anthracnose fungi and 37 ºC overnight for yeast, the MIC

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value, defined as the lowest concentration of the tested compound that causes complete

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growth inhibition, was determined. Following incubation, the growth inhibition of yeast was

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monitored using a Wallac 1420 Victor2 microplate reader (Perkin Elmer, Turku, Finland).

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Blank medium was used as the sterility control. DMSO alone at the same concentration was

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employed as a negative control. In addition, amphotericin B (Biolab, Gaithersburg, MD) was

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included in the test as a positive control. All experiments were performed in triplicate.

After

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RESULTS AND DISCUSSION

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Characterization of Actinokineospora bangkokensis strain 44EHWT. Strain 44EHWT

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was isolated from rhizospheric soil under an elephant ear plant (Colocasia esculenta) in

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Bangkok, Thailand. The colonies of strain 44EHWT are yellow in colour with white aerial

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mycelium (Figure 1A). Rod-shaped spores with a smooth surface were arranged in chains on

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aerial mycelia as shown in the scanning electron micrograph (Figure 1B). The phylogenetic

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analysis of 16S rRNA gene indicated that strain 44EHWT (GenBank accession No.

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JQ922512) also formed a clade with the closely related species in the genus

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Actinokineospora, A. enzanensis NBRC 16517T (Figure 1C).

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analysis of strain 44EHWT is currently underway.

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sequences from the genus Actinokineospora are available [A. enzanensis DSM 44649T (ID

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188797) (8,119,858 bp, GC content 70.8%), A. inagensis DSM 44258T (ID 174972)

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(7,278,759 bp, GC content 70.2%), and A. spheciospongia EG49T (ID 224112) (7,529,476

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bp, GC content 72.8%)].36 Additionally, 36 gene clusters for putative secondary metabolite

Draft genome sequence

To date, only three draft genome



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biosynthesis in A. spheciospongiae EG49T were predicted using antiSMASH.37 Besides

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being new species with both antibacterial and antifungal properties, it might contain novel

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gene clusters for the biosynthesis of uncharacterized secondary metabolites.

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Preliminary screening of antimicrobial activity from the crude extracts prepared

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from cells grown in four media. Previous studies have demonstrated that a single strain has

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the potential to produce diverse compounds when grown under various cultural parameters,

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including different medium preparations and different temperatures.

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referred to as OSMAC (one strain-many compounds).38 Several types of culture media (HA,

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NL5, MS, and SG) were used to encourage the production of diverse secondary bioactive

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metabolites from strain 44EHWT. The extracts from both supernatants and cell pellets of

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cultures grown in HA, MS and SG medium showed inhibitory activity against the growth of

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C. gloeosporioides DOA d0762 and C. parapsilosis DMKU 434. In contrast, antibacterial

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activity against B. subtilis ATCC 6051 was obtained from extracts from the supernatant and

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cell pellet of cells grown in NL5 (data not shown). Extracts from cell culture grown in HA

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were found to display potent activity against the growth of anthracnose fungi Colletotrichum

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gloeosporioides DOA d0762, C. gloeosporioides DOA c1060 and C. capsici DOA c1511

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(Figure 2).

This approach is

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In this study, liquid chromatography coupled mass spectroscopy (LC-MS) was used to

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compare isolated molecules with Prof. Hans-Peter Fiedler’s in-house compound library

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database (University of Tübingen, Germany).39

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previously identified metabolites from further investigation.

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compound with a mass of 754 Da was identified from extracts of cells cultured in HA media.

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The UV chromophore of tR at about 19 min (Figure 2D) is related to pentamycin or

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fungichromin antibiotics with a mass of 670 Da. However, no known polyene antibiotics

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with a mass of 754 Da have been described. Based on the number of conjugated double

This search allowed for the exclusion of


A putative new polyene

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bonds in the lactone ring determined from the UV-spectra, classification of members within

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the polyene class could be determined as triene, tetraene, pentaene, hexaene, heptaene or

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octaene polyenes.40

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Isolation and purification of antifungal polyene compounds. To purify putative new

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antifungal compounds, a large scale extraction (12 L) from media HA was performed and 1.7

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g of material was obtained. Based on bioactivity-guided purification, compound 1 and

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compound 2 (11.4 and 10.1 mg) were purified using semi-preparative HPLC and

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subsequently subjected to Sephadex-LH20 column chromatography to obtain compounds

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with the good purity. Peaks 1, 2 and 3 were collected based on HPLC profiles (Figure 3A)

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and their antifungal activity was monitored. Peak 2 was a major constituent from the HA

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culture broth (Figure 3A). Antifungal activity against C. gloeosporioides DoA c1060 was

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observed in peak 2 (compound 1) and peak 3 (compound 2) (Figure 3B). From the bioassay

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results, compound 2 displayed stronger activity against C. gloeosporioides DoA c1060 than

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compound 1 (Figure 3B).

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Structure elucidation of thailandins A, 1, and B, 2. The structures of 1 and 2 (Figure

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4A) were determined using data from MS, 1D and 2D NMR techniques (Table 1). Following

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LC-UV-MS analysis of the extracts a similarity search using Fiedler’s database revealed that

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the two compounds show similar UV spectra to fungichromin,39 indicating 1 and 2 to be

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members of the group of pentaene antibiotics. High resolution ESI-MS spectra of thailandins

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A, 1, and B, 2, led to determination of their molecular formulas as C39H62O14 and C33H52O10,

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respectively, based on their [M+H]+ ions. The

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atoms, and HSQC analysis indicates 2 quarternary, 25 methine, 8 methylene and 4 methyl

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carbons. Detailed analysis gave 9 double bond methine and one quarternary double bond

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carbon signal representing the presence of 5 double bonds. Moreover one ester or lactone

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functionality is shown by a signal at δC 173.1 ppm. The core structure is a 28 membered

13

C NMR spectrum of 1 shows 39 carbon



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macrocyclic lactone ring, which is decorated by two methyl groups and 6 free hydroxyl

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groups. The composition of this macrolactone ring was established from 2D NMR analysis

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(COSY, HMBC and H2BC) (Figure 5). The macrolactone ring contains a pentaene chain

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showing one quarternary double bond carbon (δC 134.3) and nine methine carbons. The

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pentaene chain position is C-16 to C-25 proven from HMBC correlations C-16/H-15, C15/-

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17H and C-25/16-H, C-25/27-H, position C-16 is methyl substituted deduced from HMBC

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correlation C-16/29-H3 and C-29/H-15. The double bonds were all identified to be in E

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configuration based on ROESY signals 29-H3/18-H, 17-H/19-H, 18-H/20-H, 23-H/25-H and

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comparison of the chemical shifts to those of fungichromin which show good agreement.41

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The molecular formula of 1 (C39H62O14) led to 9 double bond equivalents, 5 double bonds,

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the lactone, the ring closure of the macrocyclic ring and the attached sugar gave 8 double

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bond equivalents. As there are no additional signals for additional double bonds or carbonyls

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in the NMR spectra, this led to the assumption of an additional ring formation giving the

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ninth double bond equivalent. In analogy to amphothericin structures we deduce it to have a

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ring formation via a C9-O-C13 ether bond.42

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Using Kishi’s database method43 the relative configuratuion of 1,3,5-trisubstituted acyclic

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compounds like 1,3,5-triols can be determined based on the characteristic chemical shift of

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the carbon atom in the middle position, which is used for determination of novonestmycins,44

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desertomycin and oasomycin,44 tetrafibricin,45 or langkolide46 as examples.

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contains a 1,3,5-triol system at C-3, C-5 and C-7 which can be defined as syn/syn-

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configuration based on the carbon chemical shift of C-5 at δC 69.8 ppm. This is not possible

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using ROESY couplings due to strong signal overlap of 5-H, 7-H and 9-H. 2-H shows two

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axial-axial couplings of J2-H,3-H = 11.2 and J2-H,1'-Ha = 8.0 Hz which indicates 2-H and 3-H to

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be axial and 3-OH to be equatorial. ROESY signal 9-H/11-H indicates these two protons to

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be axial. The ROESY signal 13-H/14-H together with the coupling constant J12-H/13-H = 10.4


Thailandins

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Hz also show 13-H to be axial. This is supported from axial-axial coupling J14-H/15-H = 9.0

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Hz. Moreover 26-H and 27-H are both axial oriented proven by the axial-axial coupling J26-

299

H/27-H

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configuration of the macrolactone ring of thailandin A, 1, is determined as shown in Figure 6.

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Six carbon atoms were assigned to a sugar moiety, which is attached to a macrocyclic ring

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system with an additional butyl side chain. The butyl group is attached at position C-2

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established from HMBC correlations C-1/1'-Hb, C-2/1'-Hb, C-1'/2-H and H2BC correlation C-

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1'/2-H and C-2/1'-Hb. The composition of the sugar was determined as 6-desoxyhexose by

305

use of COSY, H2BC and HMBC.

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establishing these protons to be equatorial proving this sugar to be an α-sugar. 3''-H, 4''-H

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and 5''-H show axial-axial couplings indicating these protons to be in axial positions.

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Therefore this sugar can be assigned as α-rhamnose. The rhamnose is attached at position C-

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15 as determined from HMBC signals C-15/1''-H and C-1''/15-H.

= 8.0 Hz which is supported from ROESY signal 26-H/28-H3. Thus the relative

The 1''-H and 2''-H show no axial-axial couplings

310

Thailandin B, 2, is not glycosylated and shows a free hydroxyl group at position C-15,

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which is the only difference in this molecule compared to thailandin A, 1. In comparison to

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fungichromin (Figure 4B), the lactone ring shows the same pentaene unit and the same

313

hydroxylation pattern, but our compounds show a butyl group at position C-2, an ether bridge

314

C9-O-C13 and thailandin A, 1, is glycosylated at position C-15.47

315

In vitro antifungal activities of thailandins A, 1, and B, 2. Both compounds display

316

antifungal activities against anthracnose fungi and yeast with similar MIC values at 16-32

317

µg/mL. The MICs for each compound are shown in Table 2.

318

fungichromin has been previously reported to be 72 µg/mL14, indicating that thailandins A

319

and B exhibit stronger antifungal activity, based on their lower MIC values. The mode of

320

action of polyene and its derivatives is interaction with membrane sterols. The interaction of

321

polyene compounds results in the formation of aqueous pores, with the polyene hydroxyl


The MIC value for


Page 14 of 32

14

322

residues facing inward, leading to altered membrane permeability, leakage of vital

323

cytoplasmic components, and death of the organism.48 Thailandins A, 1, and B, 2, are also

324

likely to target ergosterol in a manner similar to other polyene antibiotics. However, further

325

studies are necessary to establish the mode of action of thailandins A, 1, and B, 2, produced

326

by strain 44EHWT.

327

In summary, the results provided here suggested that this new Actinokineospora species,

328

44EHWT, is a promising source of new bioactive molecules that have the potential to be

329

exploited as biocontrol agents against Colletotrichum spp.

330 331

ASSOCIATED CONTENT

332

Supporting Information

333

The Supporting Information is available free of charge at the ACS Publications website at

334

http://pubs.acs.org. Isolation procedure, NMR data and spectra (1D and 2D), MS spectrum (PDF).

335 336 337

AUTHOR INFORMATION

338

Corresponding Author

339

*Tel.: +66-20-15927. Fax: +66-20-15926. E-mail: [email protected].

340

Funding

341

This study was funded by a research grant from National Research Council of Thailand and

342

also partially supported by Thailand Research Fund through the Royal Golden Jubilee Ph.D.

343

Program (Grant No. Ph.D/0314/2551) to WP and BI.

344

Notes

345

All authors declare that there is no conflict of interest.

346


Page 15 of 32


15

347

ACKNOWLEDGEMENTS

348

We thank Mr. Andreas Kulik (University of Tübingen, Germany) for LC-MS analysis and

349

database search in Prof. Dr. Hans-Peter Fiedler’s in-house natural compound database. We

350

thank Dr. Sven Meyer (Bruker Daltonik GmbH, Bremen, Germany) for high-resolution mass

351

analysis and Mrs. Elisabeth Welle (Albert-Ludwigs University of Freiburg, Germany) for

352

technical support. The authors are grateful to Dr. Laran T. Jensen (Department of

353

Biochemistry, Faculty of Science, Mahidol University) for critical proofreading of the

354

manuscript.

355 356

ABBREVIATIONS USED

357

COSY, correlation spectroscopy; H2BC, heteronuclear 2-bond correlation; HMBC, 1H-

358

detected heteronuclear multiple bond correlation; MIC, minimum inhibitory concentration;

359

ROESY, rotating frame nuclear overhauser effect spectroscopy;

360



Page 16 of 32

16

361

REFERENCES

362

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364

2000.

365 366 367 368

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Streptomyces strains. ChemBioChem. 2015, DOI: 10.1002./cbic.201500297

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minimum inhibitory concentrations of antifungal agents for conidia forming moulds.

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secondary metabolites by HPLC and UV-visible absorbance libraries. Nat. Prod. Lett. 1993,

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Streptomyces cellulosae. J. Am. Chem. Soc. 1988, 110, 2938–2945.

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Jereczek, E.; Adlercreuz, H. Chemical studies with amphotericin B III. The complete

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structure of the antibiotic. Tetrahedron Lett. 1970, 45, 3909-3914.

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(44) Wan, Z.; Fang, W.; Shi, L.; Wang, K.; Zhang Y.; Zhang, Z.; Wu, Z.; Yang, Z.; Gu, Y.

483

Novonestmycins A and B, two new 32-membered bioactive macrolides from Streptomyces

484

phytohabitans HBERC-20821. J. Antibiot. 2015, 68, 185-190.

485

(45) Kobayashi, Y.; Tan, C.-H.; Kishi, Y.; Stereochemical assignment of the C21-C38

486

portion of the desertomycin/oasomycin class of natural products by using universal NMR

487

databses: prediction. Angew. Chem. Int. Ed. 2000, 29, 4279-4281.

488 489

(46) Kobayashi, Y.; Czechtizky, W.; Kishi, Y. Complete stereochemistry of tetrafibricin. Org. Lett. 2003, 5, 93-96.

490

(47) Helaly, S.; Kulik, A.; Zinecker, H.; Ramachandran, K.; Tan, G. Y. A.; Imhoff, J.;

491

Süssmuth, R.; Fiedler, H.-P.; Sabratnam, V. Langkolide, a 32-membered macrolactone

492

antibiotic produced by Streptomyces sp. Acta 3062. J. Nat. Prod. 2012, 75, 1018-1024.

493

(48) Ghannoum, M. A.; Rice, L. B. Antifungal agents: mode of action, mechanisms of

494

resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol.

495

Rev. 1999, 501–517.

496 497



Page 22 of 32

22

498

Legends to the Figures

499 500

Figure 1. (A) Colony morphology of strain 44EHWT on ISP medium 4 incubated at 27 oC

501

for two weeks. (B) Scanning electron micrograph of strain 44EHWT grown on ISP medium 4

502

for two weeks at 27 ºC. Bar, 5 µm. (C) Neighbour-joining phylogenetic tree based on 16S

503

rRNA gene sequences of strain 44EHWT and members of the genus Actinokineospora.

504

Bootstrap values above 50% (percentages of 1000 replications) are shown. Solid circles

505

indicate branches that are also recovered in the maximum-parsimony tree and the minimum-

506

evolution tree. Bar, 0.01 substitutions per nucleotide position; *, strains with complete

507

genome sequence; **, antibiotic producing strains

508

Figure 2. Antifungal activity of crude extracts from strain 44EHWT cultured in HA against

509

conidial suspension of Colletotrichum gloeosporioides DoA d0762 (A), C. gloeosporioides

510

DoA c1060 (B), and C. capsici DoA c1511 (C). The results were observed at day 5. (D)

511

UV/Vis spectrum of the main HPLC peak at tR 19.55 min showing absorption maxima at 325,

512

320 and 358 nm.

513

Figure 3. (A) HPLC profiles of 44EHWT crude extract and purified compounds 1 and 2. (B)

514

Antifungal activity of purified peaks 1, 2 and 3 from HA crude extract against conidial

515

suspension of C. gloeosporioides DoA c1060. The amount of purified compounds was 100

516

µg/disk.

517

Figure 4. (A) Chemical structures of thailandin A, 1, and B, 2, and (B) fungichromin.

518 519

Figure 5. Selected 2D NMR correlations of thailandin A, 1.

520 521

Figure 6. Key ROESY correlations and key coupling constants of thailandin A, 1.


Page 23 of 32


23

B

A

68

C

Actinokineospora terrae IFO 15668T (AB058394) Actinokineospora cianjurensis ID03-0810T (AB473945) Actinokineospora globicatena NRRL B-24048T (AF114798)

77 64

Actinokineospora diospyrosa NRRL B-24047T (AF114797) Actinokineospora baliensis ID03-0561T (AB447488) Actinokineospora auranticolor IFO 16518T (AB058396) Actinokineospora riparia NRRL B-16432T (AF114802)

56

Actinokineospora inagensis NRRL B-24050T (AF114799) * Actinokineospora cibodasensis ID03-0748T (AB447489) Actinokineospora spheciospongiae EG49T (GU318361)* ; ** Actinokineospora fastidiosa DSM 43855T (GQ200601)

84

Actinokineospora soli YIM 75948T (JN005785) Actinokineospora bangkokensis 44EHWT (JQ922512)**

90

Actinokineospora enzanensis IFO 16517T (AB058395) * Microbacterium lacticum IFO14135T (AB007415)

0.01

Figure 1. Intra et al., 2016



Page 24 of 32

24

* D A D 1 , 1 9 .5 5 0 (1 0 8 8

D

340

m A U 1 0 0 0 8 0 0

m A U , - ) R e f= 1 8 .0 0 0

358

325

6 0 0 4 0 0

C

2 0 0 0 3 0 0

4 0 0



5 0 0

n m

&

2 0 .0 3

Page 25 of 32


25

A DAD1 B, Sig=340

Peak 2

mAU

T

44EHW crude extract

800 600

Peak 3

400

Peak 1

200 0 5

10

15

20

25

DAD1 B, Sig=340

30

35min

Compound 1 (Peak 2)

mAU 2000 1500 1000 500 0 5

10

15

20

25

DAD1 B, Sig=340 mAU 800 600 400 200 0 5

30

35min

Compound 2 (Peak 3)

10

15

20

25

B



30

35min


Page 26 of 32

26

A

B



Page 27 of 32


27




Page 28 of 32

28



Page 29 of 32


29

Table 1. NMR Spectroscopic Data for Thailandins A, 1, and B, 2, in DMSO-d6 (δH: 600 MHz, δC: 150 MHz , Temperature = 35 °C) 1

Carbon

2

no.

δC in ppm

1

173.1

2

52.48

2.25 ddd (11.2, 8.0, 3.6)

50.4

2.25 ddd (11.1, 8.2, 3.7)

3

70.9

3.62 ddd (11.2, 9.5, 2.5)

70.8

3.62 ddd (11.1, 8.9, 2.9)

4

41.5

1.32 m

41.5

1.31 m

5

69.8

3.88 m

69.6

3.89 m

6

43.8

1.38 m

43.6

1.33 m

7

69.7

3.89 m

69.7

3.88 m

8

43.6

1.30 m

43.8

Ha: 1.41 m

δH in ppm, mult (J in Hz)

δC in ppm

δH in ppm, mult (J in Hz)

173.2

Hb: 1.30 m 9

71.0

3.87 m

71.1

3.88 m

10

42.8

Ha: 1.36 m

42.7

Ha: 1.38 m

Hb: 1.27 m

Hb: 1.28 m

11

69.2

3.78 dd (8.8, 2.8)

69.2

3.79 m

12

38.6

Ha: 1.61 m

38.7

Ha: 1.59 m

Hb: 1.35 m

Hb: 1.37 m

13

68.4

3.14 dm (10.4)

68.3

3.15 d (10.9)

14

74.8

3.63 dd (9.0, 2.0)

76.5

3.49 dd (8.6, 0.9)

15

81.9

3.75 d (9.0)

78.0

3.70 d (8.8)

16

134.3

17

130.5

6.01 d (11.0)

127.0

5.94 dd (11.8, 10.0)

18

127.3

6.48 dd (14.1, 11.0)

127.9

6.46 dd (14.4, 11.2)

19

133.9

6.33 m

132.92

6.27 m

20

132.3

6.36 m

132.4

6.34 m

21

133.0

6.35 m

132.90

6.34 m

22

131.5

6.26 m

131.6

6.26 m

23

133.5

6.36 m

132.7

6.33 m

24

129.2

6.32 m

129.1

6.32 m

25

135.1

5.96 dd (14.5, 4.8)

135.0

5.96 dd (14.4, 9.2)

26

71.3

3.95 dd (8.0, 4.8)

71.3

3.95 dd (8.3, 4.7)

27

72.9

4.65 dq (8.0, 6.4)

72.9

4.64 dq (8.5, 6.4)

138.8



Page 30 of 32

30

28

18.0

1.22 d (6.4)

18.0

1.23 d (6.4)

29

11.6

1.66 s

11.7

1.70 s

1'

28.7

Ha: 1.68 m

28.7

Ha: 1.67 m

Hb: 1.45 m 2'

31.2

Ha: 1.24 m

Hb: 1.44 m 28.8

Hb: 1.16 m

Ha: 1.28 m Hb: 1.15 m

3'

21.9

1.27 m

21.9

1.26 m

4'

13.7

0.85 t (6.8)

13.7

0.85 t (7.0)

1''

96.0

4.37 d (1.2)

2''

70.9

3.55 dd (3.2, 1.2)

3''

70.5

3.60 dd (9.6, 3.2)

4''

72.2

3.17 dd (9.6, 9.2)

5''

68.1

3.71 dq (9.2, 6.0)

6''

18.0

1.12 d (6.0)

Abbreviation: s = singlet, d = doublet, t = triplet, dd, = doublet of doublets, ddd = doublet of doublet of doublets, dq = doublet of quartets, m = multiplet


Page 31 of 32


31

Table 2. Minimum Inhibitory Concentrations (MICs) of Thailandins A, 1, and B, 2, against Anthracnose Fungi and Yeasts

MIC (µg/mL) Microorganisms Compound 1

Compound 2

Amphotericin B

Colletotrichum gloeosporioides DoA d0762

16

16

0.25

Colletotrichum gloeosporioides DoA c1060

32

16

0.25

Colletotrichum capsici DoA c1511

32

16

0.25

Candida albicans MT 2013/1

16

16

0.25

Candida parasilopsis DKMU 434

32

16

0.25

Cryptococcus neoformans MT 2013/2

16

16

0.25

Saccharomyces cerevisiae IFO10217

16

8

0.25



Page 32 of 32

32

Graphic for table of contents


Thailandins A and B, New Polyene Macrolactone ... - ACS Publications

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