Mechanistic Insights into Stereospecific Bioactivity and Dissipation of

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Agricultural and Environmental Chemistry

Mechanistic insights into stereospecific bioactivity and dissipation of chiral fungicide triticonazole in agricultural management Qing Zhang, Zhaoxian Zhang, Bowen Tang, Beibei Gao, Mingming Tian, Edmond Sanganyado, Hai-yan Shi, and MingHua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01771 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Journal of Agricultural and Food Chemistry

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Mechanistic insights into stereospecific bioactivity and

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dissipation of chiral fungicide triticonazole in agricultural

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management

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Qing Zhang †, ‡, Zhaoxian Zhang †, ‡, Bowen Tang §, Beibei Gao

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Edmond Sanganyado ┴, Haiyan Shi †, ‡, Minghua Wang †, ‡, *,

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†

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University, Nanjing 210095, P. R. China

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‡

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Application, Nanjing 210095, P. R. China

†, ‡

, Mingming Tian #,

Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural

State & Local Joint Engineering Research Center of Green Pesticide Invention and

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§

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China

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#

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Environment, Nanjing University, Nanjing 210023, P. R. China

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┴

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*Corresponding Author: E-mail: [email protected]. Phone: +86 25 84395479.

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Fax: +86 25 84395479.

College of Pharmaceutical Sciences, Xiamen University, Xiamen 361102, P. R.

State Key Laboratory of Pollution Control & Resource Reuse, School of the

Marine Biology Institute, Shantou University, Shantou 515063, P. R. China

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ACS Paragon Plus Environment


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ABSTRACT:

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Research interest on chiral pesticides has increased probably because

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enantiomers often exhibit different environmental fate and toxicity. An investigation

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into the enantiomer-specific bioactivity of chiral triticonazole enantiomers in

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agricultural systems revealed intriguing experimental and theoretical evidence. For

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nine of the phytopathogens studied (Rhizoctonia solani, Fusarium verticillioide,

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Botrytis cinerea (strawberry and tomato), Rhizoctonia cereali, Alternaria solani,

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Gibberella zeae, Sclerotinia sclerotiorum, Pyricularia grisea), the fungicidal activity

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data showed (R)-triticonazole was 3.11-82.89 times more potent than the

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(S)-enantiomer. Furthermore, (R)-triticonazole inhibited ergosterol biosynthesis and

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cell membrane synthesis more 1.80-7.34 times higher than its antipode. Homology

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modeling and molecular docking studies suggested the distinct bioactivities of the

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enantiomers of triticonazole were probably due to their different binding modes and

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affinities to CYP51b. However, field studies demonstrated that (S)-triticonazole was

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more persistent than (R)-triticonazole in fruits and vegetables. The results showed that

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application of pure (R)-triticonazole, with its high bioactivity and relatively low

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resistance risk, instead of the racemate in agricultural management would reduce the

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application dosage required to eliminate carcinogenic mycotoxins and any

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environmental risks associated with this fungicide, yielding benefits in food safety

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and environmental protection.

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KEYWORDS: chiral triticonazole; enantioselective bioactivities; stereoselective

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dissipation; agricultural management; environmental protection

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INTRODUCTION

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Chirality is a ubiquitous concept in many scientific fields, including biology,

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medicine, environmental science, chemistry, and agriculture 1. Numerous chiral

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molecules generally interact with enzymes and biological receptors in an

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enantioselective fashion 2. The tertiary stereocenters of chiral enantiomers are

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important core structures which affect the molecule’s biological and pharmacological

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properties

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binding to one of the enantiomers in biological systems, which might lead to distinct

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therapeutic or adverse effects

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variety of plant pathogens for the purpose of crop protection. This class of fungicides

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contains one or two chiral centers, but they are primarily applied as mixtures of

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racemates for chemosynthetic and economic reasons. These compounds are known to

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inhibit a cytochrome P450-dependent lanosterol 14α-demethylase (CYP51) via a

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mechanism in which the heterocyclic nitrogen atom binds to the heme iron atom in

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the binding site of the enzyme 8. It has been shown that the enantiomers of triazole

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fungicides display dramatically different toxicities and biological activities in chiral

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environments

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systematic biological evaluation of all enantiomers for pharmaceutical candidates

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Therefore, it is necessary to understand the bioactivity of each pure enantiomer for

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chiral triazole fungicides and to study the molecular mechanisms of their

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stereospecific bioactivity to gain a better understanding of plant diseases 11, 12.

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3-6

. Chiral macromolecules might exhibit preferential metabolism of or

9, 10

2, 7

. Triazole fungicides are widely used to combat a

. Additionally, the US Food and Drug Administration requires a 2, 4

.

Plant pathogens cause extensive annual yield losses of staple crops worldwide 13. 3



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In addition, some of these pathogens not only reduce the yield and quality of infected

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grain, but also produce a number of mycotoxins, such as deoxynivalenol and

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zearalenone

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mammals and negatively impact human health. Triazole fungicides are well known

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for their excellent antifungal activity and relatively low resistance risk compared to

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other commonly used fungicides, and they are thus considered the mainstay of

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modern fungicides in agrochemical applications 15. Studies so far have shown that the

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degradation of chiral triazole fungicides is commonly enantioselective in soil

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For example, the microbial transformation of triadimefon to triadimenol was

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enantioselective, with the (S)-enantiomer being preferentially transformed in soils 18.

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In addition, (R)-flutriafol exhibited a longer elimination half-life than its antipode,

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resulting in the accumulation of the (S)-isomer in rabbits

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enantioselective plant uptake and accumulation of chiral triazole fungicides in crops 9,

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10

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chiral triazole fungicides in agricultural systems is lacking.

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. Mycotoxins may cause adverse toxicological effects in exposed

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9, 16-18

.

. Little is known about

. Although these compounds are widely used, vital stereospecific knowledge of the

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To better understand the enantioselective bioactivity and degradation of chiral

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triazole fungicides in agriculture, one of the broad-spectrum, systemic chiral triazole

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fungicides triticonazole was investigated at length. In the present study,

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stereoselectivity was evaluated during biodegradation in four kinds of fruits and

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vegetables by applying racemic triticonazole in field conditions. Individual

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enantiomers were used for characterizing their stereospecific bioactivity to nine

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common plant pathogens in lab conditions. Furthermore, structural analysis and 4


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computer modeling technology was used to investigate how the selective interactions

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of triticonazole enantiomers with cytochrome P450 14α-sterol demethylases (CYP51)

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in plant pathogens lead to their enantioselective bioactivity. The results of this

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research will be helpful in elucidating the molecular mechanisms of the

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enantioselective inhibition of fungal cell-wall synthesis in plant pathogens targeted by

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chiral triazole fungicides possessing multiple chiral centers. This knowledge will also

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establish a more complete understanding of the different therapeutic or adverse effects

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which characterize each enantiomer. The insight gained from this study will be helpful

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in promoting the application of bioactive enantiomers while reducing the unwanted

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effects of invalid enantiomers as high-risk pollutants in agricultural management.

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MATERIALS AND METHODS

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Chemicals, phytopathogens, and culture conditions. Racemic triticonazole

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(98.2% purity), rac-(E)-5-[ (4-chlorophenyl) methylidene]-2,2-dimethyl-1-(1H-1,2,4-

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triazol-1-ylmethyl) cyclopentanol (Fig. S1), and ergosterol (98.0% purity) were

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purchased from the China Standard Material Center (Beijing, China). Two pure

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enantiomers of triticonazole (purity ≥99.0%) were prepared from the Shanghai

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Chiralway Biotech Co., Ltd (Shanghai, China). The ee value of (R)-triticonazole and

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(S)-triticonazole were 99.72% and 99.52%, respectively. The cellulose tris

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(3-chloro-4-methylphenylcarbamate) chromatographic column (Lux Cellulose-2,

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Phenomenex, Torrance, USA) packed with 3-μm particles (250 mm×4.6 mm i.d.) was

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used for the chiral separation of triticonazole. For achiral analysis, a C18 column

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(150×4.6 mm, 4.6 µm) was used. Sample extraction was conducted using a Cleanert 5



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Florisil-SPE cartridges (500 mg, 6 mL) obtained from Agela Technologies (Tianjin,

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China). All chromatographic solutions (acetonitrile, methanol, and n-hexane) were

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purchased from Tedia (Fairfield, USA). Analytical grade reagents (sodium chloride,

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potassium hydroxide, anhydrous sodium sulfate, and anhydrous magnesium sulfate)

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were purchased from commercial sources. Purified water was prepared using a

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MUL-9000 water purification system (Nanjing Zongxin Water Equipment Co. Ltd,

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China). The stock solution of (R)-, (S), and rac-triticonazole were each stored at

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-20 °C, and the two enantiopure standards were found to be stable over six months.

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Nine of the test phytopathogens (Rhizoctonia solani, Fusarium verticillioide,

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Botrytis cinerea (strawberry and tomato), Rhizoctonia cereali, Alternaria solani,

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Gibberella zeae, Sclerotinia sclerotiorum, Pyricularia grisea) were obtained from the

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Key Laboratory of Pesticides, Nanjing Agricultural University (Nanjing, China).The

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Potato Dextrose Agar (PDA) consisted of 200 g of potato, 20 g of agar, and 20 g of

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dextrose for every 1 L of distilled water, and the Potato Dextrose (PD) was based on

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PDA without the agar.

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Bioassay of fungicidal activities. Rac-triticonazole and its enantiomers were

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prepared in acetone solvent. A high concentration of (R)-, (S)- and rac-triticonazole

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were added into separate unsolidified PDA after the autoclaved media had reached a

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temperature of approximately 45-50 °C. Both of enantiomers are not interconversion

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and racemization in PDA solution. To avoid the influence of acetone solvent on the

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growth of phytopathogens, the concentration of acetone in the PDA medium was kept

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below 0.1 ml L-1. Mycelial plugs (5 mm in diameter) from the margins of actively 6


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growing colonies were transferred to solidified PDA plates which contained different

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concentrations of (R), (S), and rac-triticonazole in aseptic conditions. The same

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concentration of acetone was added into plates as a positive control. Each isolate was

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incubated in the dark at 25 °C with three replicates. Since, the growth rate of each

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phytopathogen was different, all colony diameters were determined when the control

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colony diameter had reached approximately 7 cm. The percent growth inhibition was

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calculated according to the following equation 9, 10:

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Inhibition (%) = (colony diameter in the control - colony diameter in the treatment) ×

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100 / (colony diameter in the control-5 mm)

(1)

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The median effective concentrations (EC50) of (R), (S), and rac-triticonazole for

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the nine phytopathogens were calculated by a linear regression of the colony diameter

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to a log of the transformed chemical concentration using DPS data-processing

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software.

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Extraction and analysis of ergosterol in phytopathogens. Mycelia of nine

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phytopathogens were cultured in 1000-mL flasks (15 mycelial plugs per flask)

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containing 400 mL of liquid potato dextrose (PD) in a rotary shaker (160 rpm) at 25°C,

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whereas the mycelial plugs (5 mm diameter) were harvested from actively growing

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colonies. When the mycelial plugs grew to the appropriate size after several days, the

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different triticonazole enantiomers were separately added into liquid potato dextrose

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(PD). The spiking concentrations of (R), (S), and rac-triticonazole were close to the

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EC90 values for each pathogen and described in the Table S1. The liquid potato

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dextrose without triticonazole served as a control and all treatments were in triplicate. 7



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After a 72 h incubation, ergosterol was extracted from each phytopathogen according

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to previous research with some modifications, and then ergosterol concentrations were

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determined using high performance liquid chromatography (HPLC)

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mycelia were collected by filtration and washed with sterile water several times, and

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the mycelia were dried in a vacuum freeze drier. Approximately 0.5 g of dry mycelia

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were broken with liquid nitrogen and subsequently suspended in 20 mL of freshly

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prepared methanolic KOH (10 %, w/v) solvent. The mixtures was homogenized for 1

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min, sonicated for 10 min, and then saponified at 80 °C for 90 minutes. To extract

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ergosterol, a mixture of 5 mL distilled water and 10 mL n-hexane was added followed

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by vigorous vortexing for 1 min. The samples were then centrifuged for 5 min at 3000

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rpm. The ergosterol extraction procedure was repeated three times, then the n-hexane

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supernatants from each extraction stage were pooled, evaporated to dryness, and then

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re-dissolved in 10 mL of methanol-water (98:2, v/v). Before analysis, the samples

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were filtered through a 0.22-µm filter membrane (Tengda, Tianjin, China). HPLC

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analyses were conducted on an Agilent 1200 HPLC system (Agilent, USA) with a

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ultraviolet detector. The separation of ergosterol was accomplished on a C18 column

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(150×4.6 mm, 4.6 µm). Isocratic elution was conducted using a mobile phase

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comprising of methanol and water (98:2, v/v) at a flow rate of 1.0 mL min-1. The

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injection volume was 20 μL and the detection wavelength was set at 282 nm.

20-23

. Briefly,

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Cell membrane permeability of phytopathogens. Prior to collection of plant

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pathogen mycelia, the same concentration of triticonazole enantiomers as used in the

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last experiment was separately added into liquid PD and incubated for 36 h (Table S1). 8


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After incubation, the mycelia were filtered on a double gauze, and thoroughly washed

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using distilled water. The mycelia (0.500 g) were suspended in separate centrifuge

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tubes containing 20 mL of double-distilled water 24. All treatments were performed in

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triplicate at each time point. Then, the electrical conductivity in this system was

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assessed by determining the cell membrane permeability at different time intervals

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using a conductivity meter (CON510 Eutech/Oakton, Singapore). Finally, the

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centrifuge tubes were put into boiling water for 5 min to allow for thorough

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breakdown of the cell membrane, and final conductivity was determined. The relative

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conductivity of the various mycelia was calculated according to the following

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equation:

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Relative conductivity (%) = Conductivity at different time points / Final conductivity × 100

(2)

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Field experiments. Four kinds of vegetables (tomatoes, cucumbers, spinach,

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Chinese cabbage) were prepared under field conditions in Nanjing, China. An

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experimental field with no application history of triazole fungicide was divided into

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several plots, each with an area of 30 m2 and insulated with a buffer zone. When the

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vegetables are grown in appropriate stages according to the BBCH-scale, triticonazole

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(2.5%, FS) was used as a foliar treatment with an application rate of approximately

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400 g a.i. ha-1 (two times the recommended dose). Besides triticonazole, no other

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triazole fungicides were applied to the crops during the field trials. Each field

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experimental treatment was set up in triplicate for the dissipation rate study.

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Representative vegetable samples were collected at different periods of time after 9



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spraying. All samples were separately ground and stored in glass containers at -20 °C.

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Extraction and analysis for vegetables. A 20 g aliquot of each vegetable

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sample was extracted using 50 mL of ethyl acetate in a 100 mL Teflon centrifuge tube.

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The extraction and cleanup procedures for chiral triticonazole were based on a

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previous method with modifications

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min followed by sonication for 15 min. After that, triticonazole was salted out from

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the aqueous phase using 2.0 g of sodium chloride. The extraction mixture was

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vigorously vortexed for 1 min and then centrifuged at 4,000 rpm for 5 min. The ethyl

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acetate supernatant (~25-mL) was collected and evaporated prior to sample

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purification using Florisil-SPE. Following purification, the sample was filtered

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through a 0.22 μm filter before HPLC analysis. The chiral separation and

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determination of enantiomers were according the previous established methods25.

206 207 208

25

. Briefly, the samples were homogenized for 3

Enantiomeric fraction (EF) described the enantiomeric composition of chiral compounds via the following equation 9: EF = R/(R + S)

(3)

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where (S)-(+)-triticonazole was eluted first, followed by (R)-(-)-triticonazole on the

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chiral chromatographic column. The R and S in this equation was the concentration of

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(R) and (S) enantiomers, respectively.

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Homology modeling and molecular docking. Cytochrome P450 14α-sterol

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demethylases (CYP51) are essential enzymes in the process of sterol biosynthesis in

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phytopathogens [26]. Triazole fungicides used in treatment of topical and systemic

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mycoses function as inhibitors of CYP51, thus it is important to further explore the 10


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mechanisms of triticonazole’s enantioselective bioactivity. The primary sequences of

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nine phytopathogens are very similar to those of Aspergillus fumigatus, with the

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sequences of six plant pathogens being over 58.5% similar, and the remaining three

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being over 22.2% similar (Table S2). Therefore, the crystal structure (PDB: 4UYM)

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of template proteins (CYP51B) for Aspergillus fumigatus was used for homology

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modeling. Three-dimensional models of CYP51B in six plant pathogens with high

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sequence similarity (Botrytis cinerea (strawberry and tomato), Gibberella zeae,

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Fusarium verticillioide, Sclerotinia sclerotiorum, Pyricularia grisea) were built based

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on the aligned sequences by homology modeling using the Prime application in

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Schrodinger Maestro Suite 2015 (Prime, version 3.9, Schrodinger, LLC, New York,

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NY, 2015). The initial three-dimensional structural models were selected and

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energy-minimized for further refinement using the OPLS 2005 force field in

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Schrodinger Prime. The validated models were optimized using the Protein

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Preparation Wizard in Schrodinger Maestro Suite 2015 prior to simulated docking

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with chiral triticonazole.

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The docking between the CYP51b models from five types of plant pathogens and

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chiral triticonazole enantiomers was performed using the Glide approach integrated in

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Maestro 10.1 (Schrodinger, LLC, 2015), with standard precision mode. The binding

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site was centered on the co-factor HEM (heme) and the metal-ligand interactions were

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set as docking constraints in the Glide grid generation process, in which the remaining

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settings used default parameters. Two enantiomers of chiral triticonazole were

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separately docked into the binding site after preparation using LigPrep with the 11



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docking protocol. Glide Score (GScore) as a scoring function is used to describe each

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docking complex conformation, and the values generated are negative values. The

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best-ranked complex conformation of each enantiomer by GScore was selected to

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analyze the different binding modes with CYP51b for chiral triticonazole. A higher

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absolute docking value of GScore means that the docking between ligand and receptor

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is more stable 27, 28.

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RESULTS AND DISCUSSION

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Enantioselective

bioactivity

in

phytopathogens.

The

stereochemical

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bioactivity of chiral triticonazole for nine phytopathogens was determined using a

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biological assay based on the inhibition of bacteriostatic circle method. The racemic

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mixture, rac-triticonazole, exhibited antifungal activity against all phytopathogens

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with EC50 values of less than 1.00 mg kg-1, exhibited antifungal activity against all

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phytopathogens with EC50 values less than 1.00mg kg-1, except for Pyriculariagrisea

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where an EC50 value of 2.16 mg kg-1 was obtained (Table 1). Furthermore, the

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antifungal activity of rac-triticonazole against Botrytis cinereal varied between

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different host plant species. For example, based on EC50 values, rac-triticonazole was

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2.33 times more effective in Botrytis cinereal infected strawberry plants than tomato.

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As shown in Table 1, the two enantiomers of triticonazole possessed different

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fungicidal activities and the order of potency was (R)-triticonazole > rac-triticonazole >

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(S)-triticonazole in all bioassays. It was observed (R)-triticonazole was 3.11-82.89

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times more active than the (S)-enantiomer. However, enantioselective bioactivity was

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low against Pyriculariagrisea with the (R)-enantiomer only 3.11 times more potent 12


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than its antipode. In addition, the (R)-enantiomer was significantly more bioactivity

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against Fusarium verticillioide and Rhizoctoniasolani with EC50 values 43.65-82.89

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times higher than the (S)-enantiomer. Thus, the antifungal activity of triticonazole

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may be attributed primarily to the (R)-enantiomer. The EC50 values of triticonazole

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from the experiment showed in eight phytopathogens (Rhizoctoniasolani, Fusarium

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verticillioide, Botrytis cinerea (strawberry and tomato), Rhizoctoniacereali,

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Alternaria

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contributed approximately 92.07-98.81% of the activity against plant pathogens in the

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racemate. In previous studies on triazole fungicides, a 24.2-fold and 1000-fold

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difference was observed between the chiral enantiomers of difenoconazole and

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paclobutrazol, respectively 9, 29. Therefore, only one of the enantiomers of current-use

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chiral fungicides may exhibit pathogenic control properties in agricultural

272

management.

solani,

Gibberellazeae,

Sclerotiniasclerotiorum),

(R)-enantiomer

273

Enantioselectivity in ergosterol biosynthesis. To further explore the underlying

274

mechanism of triticonazole’s enantioselective bioactivity on phytopathogens, the

275

inhibition of ergosterol biosynthesis by chiral enantiomers was investigated using the

276

chromatographic analysis. The concentrations of ergosterol for all treatments are

277

shown in Fig. 1. All triticonazole treatments significantly affected the ability of

278

pathogenic

279

Enantioselectivity was observed in the inhibitory effects on ergosterol biosynthesis in

280

nine phytopathogens for the two enantiomers (Table S3). Compared to the control,

281

around 25.63-77.58%, 18.23-64.27%, and 3.63-36.52% of ergosterol biosynthesis was

cells

to

synthesize

ergosterol

following

13


exposure

for

72

h.


282

inhibited by (R), (rac), and (S)-triticonazole, respectively. The inhibition of ergosterol

283

production was generally higher (1.80-7.34 times) following (R)-triticonazole

284

exposure than (S)-triticonazole against the nine pathogens. The enantioselective

285

inhibition of ergosterol production observed was in agreement with the

286

enantioselective bioactivity found in exposed phytopathogens. A positive correlation

287

(r=0.86) between the enantioselective bioactivity of the nine phytopathogens and the

288

inhibition of ergosterol biosynthesis for the triticonazole enantiomers was obtained by

289

fitting the bioactive ratio value (R/S) to the corresponding inhibition of ergosterol

290

ratio value in Fig. 2. The results suggest that the distinct bioactivities observed for

291

chiral triazole fungicides was mainly caused by the loss of the ability to synthesize

292

ergosterol in phytopathogenic cells.

293

Enantioselectivity in cell membrane permeability. Many commercial chiral

294

triazole fungicides are capable of affecting gene expression and may thus further

295

block the process of ergosterol biosynthesis

296

process for some commercial chiral triazole fungicides which are designed to

297

eliminate plant pathogens

298

substances in fungal cell membranes. Chiral triazole fungicides inhibit the formation

299

of cell membranes and ultimately the exchange of substances across the membrane,

300

resulting in cell death

301

concentrations of racemic and enantiopure triticonazole (Table S1), and the electrical

302

conductivity was detected after different exposure times. Following triticonazole

303

treatment, electrical conductivity gradually increased with increasing treatment time

20

21

. Ergosterol biosynthesis is the target

26

. This is significant since ergosterols are essential

. Nine plant pathogenic cells were treated with appropriate

14


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and finally reached equilibrium after incubating for 180 h (Fig. S2). The results show

305

that (R)-, (S)-and, rac-triticonazole differentially enhanced the electrical conductivity

306

of the pathogenic cells. However, the electrical conductivity of nine plant pathogenic

307

cells amended with (S)-triticonazole were not significantly different from the control

308

for some of the plant pathogens (i.e. Botrytis cinerea, Pyricularia grisea, Sclerotinia

309

sclerotiorum). Furthermore, (R)-triticonazole substantially increased electrical

310

conductivity by 21.72 to 76.42% under the same treatment conditions, indicating that

311

the (R)-isomer is more potent in this regard (Table 2). Therefore, the enantioselective

312

cell membrane permeability for plant pathogenic cells may offer a more

313

comprehensive insight on the mechanism of enantioselective bioactivity caused by

314

chiral triazole fungicides. Cytochrome P450 enzyme are essential catalytic enzymes in

315

fungal ergosterol biosynthesis

316

against plant pathogenic cell requires further research to be understood, especially in

317

regard to the enantioselective relationship between chiral triazole fungicides and

318

phytopathogenic CYP51b.

30

. The precise impact of chiral triazole fungicides

319

Molecular interactions between chiral triticonazole enantiomers and

320

CYP51b. The homology modeling and molecular docking techniques are performed

321

to further explain the stereoselective molecular interactions for chiral enantiomers in

322

the phytopathogens. The composition of the three dimensional active group (i.e.

323

hydroxy, benzene, and pyrazolone ring) in triticonazole enantiomers strongly affects

324

their binding affinities to CYP51b enzymes. Both enantiomers can be tightly bound to

325

the active cavities of CYP51b, and the enantiomers are shown at different distances 15



326

from the key amino acids present (Fig. 3). For five of the phytopathogens studied (i.e.

327

Botrytis cinerea, Gibberella zeae, Fusarium verticillioide, Sclerotinia sclerotiorum,

328

Pyricularia grisea), several residues are in the active site of CYP51b within a distance

329

of 4 Å and 5 Å for three-dimensional and two-dimensional representations,

330

respectively (Fig. 3). All active site residues are summarized in Table S4. These

331

residues are closely related to the observed binding affinities for triticonazole

332

enantiomers since they determine the three-dimensional conformation of the

333

enzyme-ligand interaction. The binding affinities depend on the structure of chiral

334

compounds and of the receptor proteins themselves

335

groups (i.e. hydroxy and benzene) present in chiral triticonazole easily generate

336

hydrogen bonds and Pi-Pi stacking interactions with amino acid residues in the

337

enzyme active site. In addition, the ferric ions of heme are present in the active pocket

338

of CYP51b, allowing for Pi-cation and metal coordination interactions in the presence

339

of triticonazole. Furthermore, hydrophobic effects play an important role in

340

combining the hydrophobic functional groups of triticonazole with amino acid

341

residues 33, 34.

25, 31, 32

. Some of the functional

342

For five phytopathogens with high sequence similarity, the binding activity of

343

the association between the two enantiomers and CYP51b was investigated in terms

344

of their docking energy 31, 33. The top scored protonation formed between triticonazole

345

and CYP51b was selected, since docking score includes both binding affinity and

346

tautomeric ratio 35, 36. The two enantiomers showed distinct binding modes with amino

347

acid residues in the enzyme active site (Fig. 3). The binding affinity values of the 16


Page 16 of 35

Page 17 of 35


348

(R)-enantiomer were consistently lower than those of the (S)-isomer, suggesting that

349

(R) bound strongly to CYP51b, while (S) had poor binding activity to CYP51b in

350

phytopathogens (Table S4). The different binding activity with chiral triticonazole

351

could be synthetically caused by H-bonds, Pi-Pi stacking, Pi-cation interactions, metal

352

coordination, and hydrophobic effects, which are critical for stereoselective molecular

353

recognition

354

(R)-enantiomer exhibited greater bioactivity against common plant pathogens.

355

Therefore, the distinct bioactivities of chiral enantiomers in triazole fungicides may

356

derive from variable binding affinities and binding modes to CYP51b.

37, 38

. The results were consistent with the experimental results that the

357

Enantioselective dissipation in fruits and vegetables. The change of the value

358

of EF is a powerful metric to indicate enantioselective behavior of chiral pollution in

359

biological processes, and it was used to evaluate the enantioselective dissipation of

360

chiral triticonazole in vegetables under field conditions

361

relatively high concentrations of triticonazole after the foliar spray treatment,

362

followed by a gradual decreased with time. The dissipation of triticonazole

363

enantiomers followed first order kinetics (R2 ≥ 0.927), and the half-lives for both

364

enantiomers are shown in Table S5. The half-lives of the more bioactive

365

(R)-enantiomer were consistently shorter than those of (S)-triticonazole in the fruits

366

and vegetables studied. The average EF value for triticonazole remained constant (EF

367

= 0.5) at the beginning of the foliar spray treatment (0-5d for cucumber, 0-7d for

368

Chinese cabbage and tomato, and 0-14 d for spinach) in four plants (Fig. 4), but the

369

EF value instantly increased to 0.576-0.675 in the late stages (0.576 for tomatoes, 17


21

. The vegetables contained


Page 18 of 35

370

0.609 for spinach, 0.624 for Chinese cabbage and 0.675 for cucumbers). Preferential

371

dissipation of the (R)-enantiomer resulted in relative enrichment of the (S)-enantiomer.

372

Hence, the less bioactive (S)-enantiomer is more persistent in fruits and vegetables,

373

thus humans may be exposed to it through dietary intake. Chiral switching to

374

(R)-triticonazole from the commercial racemate will probably help in crop protection

375

against pathogens while reducing potential public health risks. The enzymatic systems

376

in plants have been shown in previous studies to play an important role in the

377

stereoselective degradation, metabolism, and accumulation of chiral pollutants

378

The absence of enantioselectivity at the beginning of the foliar spray treatment may be

379

due to the fact that the system of stereoselective enzymes had limited contact with

380

chiral triticonazole during this initial period. After this initial lag period following

381

pesticide application, the transportation and distribution of triticonazole throughout

382

the plant tissues increased in fruits and vegetables. Therefore, the system of

383

stereoselective enzymes was exposed to the chiral fungicides, resulting in a significant

384

enantioselective dissipation of the enantiomers in the vegetables.

9, 39

.

385

In this present work, Cell membrane permeability, ergosterol biosynthesis, and

386

molecular interaction investigations revealed significant differences between (R)-, (S)-,

387

and rac-triticonazole. Thus, the current study demonstrated the bioactivity and

388

dissipation of chiral triticonazole in agricultural ecosystems was enantiomer-specific.

389

The different binding affinities between the individual triticonazole enantiomers and

390

receptor proteins of CYP51b demonstrated the enantioselective bioactivities of chiral

391

triazole fungicides at molecular level. In addition, although (R)-triticonazole was 18


Page 19 of 35


392

found to be more bioactive than (S)-triticonazole against nine phytopathogens,

393

(R)-triticonazole was less persistent than (S)-triticonazole in various kinds of fruits

394

and vegetables under field conditions. Therefore, chiral switching to the more

395

bioactive and less persistent (R)-triticonazole from the currently used racemate would

396

reduce the required application dosage and hence the associated environmental risks

397

while still maintaining the fungicidal efficacy. Enantioselectivity is commonly

398

observed in several aspects of environmental science (i.e. adsorption, degradation,

399

transportation, excretion and ecotoxicology) for numerous chiral pesticides. Therefore,

400

the significance of enantioselective effects for chiral agrochemicals must be

401

considered in order to improve their risk assessment and minimize dangers to public

402

health.

403

ABBREVIATIONS USED

404

PDA, potato dextrose agar; PD, potato dextrose; EC50, effective concentrations;

405

HPLC, high performance liquid chromatography; GScore, glide score; EF,

406

enantiomeric fraction.

407

AUTHOR INFORMATION

408

Corresponding Author

409

*Phone: +86 25 84395479. Fax: +86 25 84395479. E-mail: [email protected].

410

Funding

411

This study was supported by the National Key Research and Development Program of

412

China (2016YFD0200207).

413

Notes 19



414

Conflicts of interest the authors do not have any potential conflict of interest to

415

declare.

416

ASSOCIATED CONTENT

417

Supporting Information

418

Supporting materials (Fig. S1-S2 and Table S1-S5) are available free of charge via the

419

Internet at http://pubs.acs.org.

420 421

REFERENCES

422

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423 424 425

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Cuenca-Estrella, J. M.; Rodriguez-Tudela, J. L. Ergosterol biosynthesis pathway

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in Aspergillus fumigatus. Steroids. 2008, 73, 339-347.

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fludioxonil on morphological and physiological characteristics of Sclerotinia

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sclerotiorum. Pestic. Biochem. Phys. 2013, 106, 61-67.

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A.; Sauer-Eriksson, A. E.; Andersson, P. L. A structure-based virtual screening

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targeting transthyretin. Environ. Sci. Technol. 2016, 50, 11984-11993.

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Hedden, P.; James, C. S.; Lenton, J. R. Comparative activity of the enantiomers

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of triadimenol and paclobutrazol as inhibitors of fungal growth and plant sterol

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and gibberellin biosynthesis. Pest Manag. Sci. 2010, 21, 253-267.

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c-32

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biotransformation of chiral PCBs in whole poplar plants. Environ. Sci. Technol.

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2011, 45, 2308-2316.

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Diastereomers

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dibromoethyl)cyclohexane induce androgen receptor activation in the HepG2

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hormonal network and the enantioselectivity of bifenthrin in trophoblast:

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Maternal-fetal health risk of chiral pesticides. Environ. Sci. Technol. 2014, 48,

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cancer cells: chirality in cancer development. Environ. Sci. Technol. 2015, 49,

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D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.;

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Francis, P.; Shenkin, P. S. Glide: A New Approach for Rapid, Accurate Docking

540

and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem.

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[37] Iakovleva, I.; Brännström, K.; Nilsson, L.; Gharibyan, A. L.; Begum, A.; Anan, I.;

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544

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546

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549

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550

[39] Huang, H.; Zhang, S.; Lv, J.; Wen, B.; Wang, S.; Wu, T. Experimental and

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552

biotransformation of HBCD in maize roots. Environ. Sci. Technol. 2016, 50,

553

12205-12213.

554

26


Page 26 of 35

Page 27 of 35


555

Figure Legends

556

Figure 1. The enantioselective inhibition of ergosterol biosynthesis by chiral

557

triticonazole enantiomers.

558

Figure 2. The relationship between the R/S ratio value of enantioselective bioactivity

559

and the enantioselective inhibition of ergosterol.

560

Figure 3. The two-dimensional and three-dimensional enantiomer-specific binding

561

modes for triticonazole enantiomers bound to CYP51b.

562

Figure 4. Time development of EF values for the triticonazole enantiomers in

563

vegetables.

564

27



Page 28 of 35

Table 1. EC50 values of chiral triticonazole enantiomers for nine plant pathogens. R-triticonazole

Species

Rac-triticonazole

S-triticonazole

R/Sd

EC50a (mg kg-1)

R2b

Pc

EC50 (mg kg-1)

R2

P

EC50 (mg kg-1)

R2

P

Rhizoctonia solani

0.0060±0.0005

0.9497

0.0173

0.0091±0.0007

0.9516

0.0019

0.2619±0.0057

0.9873

0.0027

43.65

Fusarium verticillioide

0.0335±0.0019

0.9574

0.0105

0.1153±0.0039

0.9830

0.0026

2.7769±0.0431

0.9870

0.0018

82.89

Botrytis cinerea (strawberry) Rhizoctonia cereali

0.1190±0.0036

0.9456

0.0048

0.4307±0.0121

0.9421

0.0058

1.3825±0.0578

0.9917

0.0057

11.62

0.1632±0.0043

0.9710

0.0231

0.3485±0.0193

0.9755

0.0134

1.9580±0.0713

0.9967

0.0151

12.00

Botrytis cinerea (tomato) Alternaria solani

0.2771±0.0203

0.9916

0.0071

0.7431±0.0363

0.9847

0.0047

4.2013±0.2151

0.9448

0.0071

15.16

0.3084±0.0176

0.9847

0.0238

0.7934±0.0219

0.9887

0.0313

4.8694±0.2637

0.9921

0.0054

15.79

Gibberella zeae

0.4066±0.0183

0.9896

0.0137

0.8034±0.0605

0.9757

0.0171

6.0840±0.4179

0.9938

0.0079

14.96

Sclerotinia sclerotiorum

0.5236±0.0471

0.9900

0.0091

0.9070±0.0786

0.9910

0.0121

11.2996±1.0021

0.9704

0.0191

21.58

Pyricularia grisea

1.7661±0.0893

0.9798

0.0275

2.1607±0.1563

0.9679

0.0029

5.4854±0.4873

0.9966

0.0023

3.11

a the effective concentration that results in a 50% reduction in population growth compared to the control. b represents the correlation coefficient. c represents the probability (associated with the t-test). d the EC50 value of R-triticonazole to S-triticonazole ratio.

28


Page 29 of 35


Table 2. Inhibited cell membrane permeability (%) in plant pathogens by chiral triticonazole enantiomers.

Gibberella

Rhizoctonia

Botrytis cinerea

Rhizoctonia

Alternaria

Botrytis cinerea

Fusarium

Sclerotinia

Pyricularia

zeae

cereali

(strawberry)

solani

solani

(tomato)

verticillioide

sclerotiorum

grisea

(R)-triticonazole

30.30

24.98

45.40

37.60

52.16

76.42

21.72

73.20

69.85

(Rac)-triticonazole

26.95

21.42

42.19

33.25

48.78

73.41

17.68

69.51

63.61

(S)-triticonazole

24.15

20.07

40.38

32.25

43.89

70.32

16.95

65.54

59.73

Untreated control

21.59

15.85

37.73

31.45

41.81

67.82

15.97

63.34

57.90

Chiral enanatiomers

29



Figure 1. The enantioselective inhibition of ergosterol biosynthesis by chiral triticonazole enantiomers in nine plant pathogens.

30


Page 30 of 35

Page 31 of 35


Figure 2. The relationship between the R/S ratio value of enantioselective bioactivity and the enantioselective inhibition of ergosterol.

31



Figure 3. The two-dimensional dimensional and three-dimensional enantiomer-specific specific binding modes for pure triticonazole enantiomers bound to CYP51b in five plant pathogens verticilllioide C Botrytis cinerea,, D Sclerotinia (A Gibberella zeae, B Fusarium verticilllioide, sclerotiorum, E Pyricularia grisea) grisea with high sequence similarity.

32


Page 32 of 35

Page 33 of 35


Figure 4. Time development of EF values for the triticonazole enantiomers in spinach (A) , cucumber (B), (B) Chinese cabbage (C) and tomato (D).

33



TOC Graphic:

34


Page 34 of 35

Page 35 of 35


509x285mm (150 x 150 DPI)


Mechanistic Insights into Stereospecific Bioactivity and Dissipation of

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