Rapid and Visual Detection and Quantitation of Ethylene Released

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New Analytical Methods

Rapid and visual detection and quantification of ethylene released from ripening fruits: the new use of Grubbs catalyst Mingtai Sun, Xin Yang, Yuannian Zhang, Suhua Wang, Ming Wah Wong, Runyan Ni, and Dejian Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05874 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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Journal of Agricultural and Food Chemistry

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Rapid and Visual Detection and Quantitation of Ethylene

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Released from Ripening Fruits: the New Use of Grubbs Catalyst

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Mingtai Sun,†, ‡ Xin Yang,† Yuannian Zhang,† Suhua Wang,‡ Ming Wah Wong,† Runyan Ni§,

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Dejian Huang*,†,§

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†Food

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Singapore, 3 Science Drive 3, 117543, Singapore

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‡School

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Beijing 102206, China.

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§National

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Science and Technology Program, Department of Chemistry, National University of

of Environment and Chemical Engineering, North China Electric Power University,

University of Singapore (Suzhou) Research Institute, 377 Linquan Street, Suzhou,

Jiangsu, 215123, China.

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*Corresponding author

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Tel: 65-6516-8821. Fax: 65-6775-7895. Email: [email protected]

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ABSTRACT:

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Herein we report on fluorophore-tagged Grubbs catalysts as turn-on fluorescent probes for the

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sensitive detection and quantitation of ethylene, a plant hormone that plays a critical role in many

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phases of plant growth and fruit ripening. The ruthenium (Ru) based weakly fluorescent probes

22

were prepared handily through metathesis reaction between the first generation Grubbs catalyst

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and selected fluorophores that have high quantum yields and contain terminal vinyl groups. Upon

24

exposure to ethylene, fluorescence enhancement was observed via the release of fluorophore from

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the probe. Our probe shows an excellent limit of detection (LOD) for ethylene at 0.9 ppm in air

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and was successfully applied for monitoring ethylene released during the fruit ripening process.

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Our work opens up a new avenue of application of Grubbs catalysts for bioanalytical chemistry of

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ethylene, which is critically important in plant biology, agriculture and food industry.

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KEYWORDS: Grubbs catalyst, ethylene, fluorescent probes, olefin metathesis reaction, fruit

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ripening

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INTRODUCTION

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Ethylene is a gaseous plant hormone that plays critical roles in many phases of plant biology

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including plant growth, development, response to environmental stresses and pathogen infection1

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as well as seed germination, and fruit ripening.2-4 In food and agriculture, ethylene concentration

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has been used as a maturity index to determine the time of harvest.5 Knowing the concentration of

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ethylene is critical since ethylene activity varies with the fruit maturity and is dependent on the

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type of fruits.6 In storage room and transportation chains, the control of ethylene concentration is

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necessary to avoid the deterioration of produce that are sensitive to ethylene.7 Ethylene

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concentrations of less than 1 ppm could be effective for the ripening of certain fruits. However,

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this concentration is usually held between 10 to 200 ppm in ripening rooms depending on the type

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of fruits.1 Thus, knowing the relationship between ethylene concentration and the fruit ripening

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process is important in order to manage the harvesting, storage and transportation processes yet

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there is no convenient way to rapidly quantitate ethylene concentrations.

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Currently, quantitation of ethylene relies heavily on time-consuming and sophisticated

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traditional methods such as GC (GC-MS), photoacoustic spectroscopy, and electrochemical

47

methods. Hence, real-time measurements are highly desired to control fruit ripening.5, 8-11 In the

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past decade, great progress has been made in the chemical biology field with the help of fluorescent

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probes that are designed to be sensitive and selective towards specific biological targets including

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small molecules of importance such as gluthathione, ATP, nitric oxide, nitrogen dioxide, singlet

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oxygen, hypochlorous acid, and hydrogen sulfide.12-19 However, only limited work has been

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reported on the fluorescent probes for ethylene detection. The reported methods took advantage of

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the ability of ethylene as a ligand that could reversibly coordinate to metals particular copper (Cu(I))

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and silver (Ag(I)).20-24 However, these methods suffer from low sensitivity because ethylene is a 3 ACS Paragon Plus Environment


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rather weak ligand and require high concentration in order to effectively undergo ligand

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substitution reaction that releases the metal bound fluorophores. Therefore, a new approach is

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needed to achieve probes with high sensitivity.25 To this end, we were intrigued by the potential

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of using alkene metathesis catalyst based on ruthenium carbene complex, PhCH=RuCl2(PCy3)2

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(Ph = phenyl, Cy = cyclohexyl) the first generation Grubbs catalyst, which has found broad

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application in organic and polymer synthesis.26, 27 We envisioned that the high tolerance of Grubbs

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catalyst towards other functional groups, good air and water stability would be of potential to

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explore its application for detection of ethylene if we could design and prepare fluorophore-tagged

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Grubbs catalysts that are responsive, selective, and sensitive to ethylene.

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MATERIALS AND METHODS

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Reagents and apparatus. Chemical reagents were purchased from the commercial sources

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(Sigma-Aldrich Chemical Co., Singapore) and used directly without further purification unless

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specified. The solvents dichloromethane and THF were further treated before use by distillation

68

and dried over molecular sieves before use. 1-Vinylpyrene, 5, was synthesized according to a

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literature report.28 Compounds 1 and 3 (Figure 1) were prepared in our lab. Aqueous solutions

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were all prepared using ultrapure water (18.2 MΩ·cm) from a Millipore water purification system,

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and all glassware was cleaned with ultrapure water and then dried before use. Fluorescence

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measurement was recorded on a Fluoromax-4 spectrofluorometer (HORIBA Jobin Yvon,

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Piscataway, NJ) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). UV/Vis absorption

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was recorded on a UV-1601 UV-visible spectrophotometer (Shimadzu, Kyoto, Japan) fitted with

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a quartz cell. The FTIR spectra were obtained with a Nicolet iS10 spectrometer (Thermo Scientific,

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Madison, WI). 1H and 13C NMR spectra were recorded with an AC300 spectrometer at 300 MHz

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or an AV500 spectrometer (Bruker, Karlsruhe, Germany) at 500 MHz. The electrospray ionization 4 ACS Paragon Plus Environment

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mass spectra were obtained from an LCQ ion trap mass spectrometer (Finnigan/MAT, San Jose,

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CA) equipped with electrospray ionization (ESI) source. Thin-layer chromatography (TLC) was

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performed by using F254 silica gel 60 plates (Merck, Singapore).

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Synthesis of compound 2. To a solution of 1st generation Grubbs catalyst (39 mg, 0.046 mmol,

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1 equiv) in dry CH2Cl2 (5 mL), compound 1 (149 mg, 0.368 mmol, 8 equiv) was added at room

83

temperature. The solution was stirred for another 15 min. The solvent was removed under vacuum,

84

and the residue was repeatedly washed with acetone (3 mL) and dried in vacuo for several hours.

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Compound 2 was obtained as an orange solid. Yield: 48 mg (92%). 1H NMR (500 MHz, CDCl3)

86

δ 20.17 (s, 1H), 8.66 (s, 2H), 7.32 (d, J = 8.5 Hz, 2H), 2.69 (m, 6H), 2.57 (s, 6H), 2.35 (q, J = 7.5

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Hz, 4H), 1.93–1.70 (m, 30H), 1.49 (m, 12H), 1.35 (s, 6H), 1.31–1.18 (m, 18H), 1.02 (t, J = 7.5 Hz,

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6H). 31P NMR (202 MHz, CDCl3) δ 35.58. 13C NMR (126 MHz, CDCl3) δ 153.95, 151.95, 140.31,

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137.99, 136.46, 132.93, 131.81, 129.65, 129.47, 32.42, 32.35, 32.28, 29.67, 27.81, 27.76, 27.73,

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26.50, 26.36, 17.10, 14.64, 12.53, 11.76. Anal. Calcd for C60H93BCl2F2N2P2Ru: C, 64.05; H, 8.33;

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N, 2.49. Found: C, 64.48; H, 8.27; N, 2.42. HR-MS (ESI positive): m/z calculated for

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[C60H93BCl2F2N2P2Ru + H]+ 1124.5308; found 1124.5318.

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Synthesis of compound 4. To a solution of 1st generation Grubbs catalyst (25 mg, 0.03 mmol,

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1 equiv) in dry CH2Cl2 (5 mL), compound 3 (96 mg, 0.18 mmol, 6 equiv) was added at room

95

temperature. The solution was allowed to stir for another 15 min. The solvent was removed under

96

vacuum, and the residue was repeatedly washed with acetone (3 mL) and dried under vacuum for

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several hours. Compound 4 was obtained as a red solid. Yield: 33 mg (91%). 1H NMR (300 MHz,

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CD2Cl2) δ 19.91 (s, 1H), 8.38 (d, J = 6.8 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 3.76 (s, 2H), 3.26 (s,

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2H), 2.72–2.35 (m, 24H), 2.12 (s, 6H), 1.84–1.60 (m, 30H), 1.42 (m, 12H), 1.20 (m, 18H), 1.13–

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0.97 (m, 6H).

31P

NMR (202 MHz, CD2Cl2) δ 36.33.

13C

NMR (126 MHz, CD2Cl2) δ 206.23,



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150.00, 130.92, 129.32, 35.55, 35.06, 32.11, 32.04, 31.96, 30.52, 29.61, 27.83, 27.79, 27.75, 26.97,

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26.88, 26.53, 26.35, 26.33, 26.20, 16.98, 14.51, 14.01. Anal. Calcd for C66H105BCl2F2N4P2Ru: C,

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64.07; H, 8.05; N, 4.53. Found: C, 64.11; H, 8.09; N, 4.52. HR-MS (ESI positive): m/z calculated

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for [C66H105BCl2F2N4P2Ru + H]+ 1237.6388; found 1237.6415.

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Synthesis of compound 6. To a solution of 1st generation Grubbs catalyst (PhCH=Ru(PCy3)2Cl2,

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59 mg, 0.072 mmol, 1 equiv.) in dry CH2Cl2 (5.0 mL), 1-vinylpyrene, 5 (165 mg, 0.72 mmol, 10

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equiv) was added at room temperature. The purple solution turned to yellow immediately and was

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stirred for another 10 min. The solvent was removed under vacuum, and the residue was repeatedly

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washed with acetone (3 mL) and dried under vacuum for several hours. Compound 6 was obtained

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as a khaki solid. Yield: 57 mg (84%). 1H NMR (500 MHz, CDCl3) δ 21.85 (s, 1H), 9.89 (d, J =

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8.4 Hz, 1H), 8.96 (d, J = 9.5 Hz, 1H), 8.43 (d, J = 7.7 Hz, 1H), 8.37 (t, J = 8.6 Hz, 3H), 8.09 (d, J

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= 8.4 Hz, 1H), 8.06–7.97 (m, 2H), 2.72 (m, 6H), 1.91–1.63 (m, CyH), 1.52–1.10 (m, CyH). 13C

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NMR (126 MHz, CDCl3) δ 144.89, 132.82, 132.18, 131.47, 131.18, 128.89, 128.37, 127.04,

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126.77, 126.21, 125.75, 125.63, 125.60, 124.59, 122.54, 32.44, 32.37, 32.30, 29.81, 27.89, 26.51.

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31P

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1174, 1005, 916, 848, 732, 714, 510. Anal. Calcd for C53H76Cl2P2Ru: C, 67.21; H, 8.09. Found:

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C, 67.05; H, 8.05. ESI 911.1(100%) (M-Cl)+, 913.1(62%), 907.1(60%), 906.1 (35%).

NMR (202 MHz, CDCl3) δ 36.09. FT-IR (KBr, cm-1): 2925, 2849, 1582, 1500, 1445, 1216,

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Reaction of 1-vinylpyrene with 2nd generation Grubbs catalyst. To a solution of 2nd

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generation Grubbs catalyst (52 mg, 0.055 mmol, 1 equiv) in dry CH2Cl2 (5 mL), 1-vinylpyrene

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(125 mg,0.55 mmol, 10 equiv) was added at room temperature and was allowed to stir for another

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10 min. The solvent was removed under vacuum, and the residue was repeatedly washed with

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acetone (5 mL) and dried under vacuum for several hours. An orange solid with strong green


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fluorescence was obtained. This compound is slightly soluble in common solvents such as hexane,

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acetone, CHCl3, toluene, CH2Cl2, and DMSO.

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Determination of ethylene concentration by NMR spectroscopic analysis. To an NMR tube,

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5 μL of methyltriethoxysilane and 0.6 mL of CD2Cl2 were added. Ethylene gas was bubbled to the

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solution for 5 min carefully using a long syringe needle. The 1H NMR spectrum of the resulting

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solution was measured. The concentration of ethylene dissolved in CD2Cl2 (nethylene) was calculated

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using the following equation:

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𝑛𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 =

𝐼𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 × 𝑛𝑀𝑇𝐸𝑆 × 6 𝐼𝑀𝑇𝐸𝑆(𝑎) × 4

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where Iethylene represents the integration of ethylene peak at δ 5.43; IMTES represents the integration

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of

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methyltriethoxysilane in CD2Cl2.

methyltriethoxysilane

peak

at

δ

3.80;

nMTES

represents

the

concentration

of

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Procedures for ethylene gas sensing and other species. Generally, probe 6 was dissolved in

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CH2Cl2 to get a stock solution with the concentration at 1×10-3 M. Ethylene gas at concentrations

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of 50 ppm, 100 ppm, and 200 ppm were obtained from commercial ethylene gas cylinder (Chem-

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Gas Pte Ltd, Singapore). Then the ethylene gas was diluted to 25, 12.5, 6.25, and 2.5 ppm by

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adding corresponding ethylene gas to a round bottom flask with a certain volume, respectively. 2

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mL of these gases were injected to 2 mL of CH2Cl2 solvent in a small sealed bottle with a plastic

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gap using a syringe slowly before 10 μL of probe 6 stock solution was added. The fluorescence

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intensity of the solution was measured after 3 min by recording the fluorescence spectra in the

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range 380-600 nm using a 365-nm excitation wavelength and a 500 nm/min scan rate. For the

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control, the probe solution was prepared by adding the stock solution of the probes into 2 mL of

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CH2Cl2 solvent, and then the fluorescence was measured. SO2, NO2, H2S, CO2, and NH3 gases 7 ACS Paragon Plus Environment


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were prepared according to previous methods.29 Generally, 200 μL of these gas samples were

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bubbled into the probe solution in CH2Cl2 before 10 μL of the probe stock solution was added and

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the fluorescence spectra were recorded. For the selectivity of the probe towards ethylene solution

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in CH2Cl2, the known concentration of ethylene in CD2Cl2 was used to prepare the desired ethylene

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solution at concentrations of 5 and 10 μM. Other species including acetonitrile, ethanol, ethyl

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acetate, water, toluene, THF, 1-propanol, and 1-butanol were calculated and taken using a pipette

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to 2 mL of CH2Cl2 solvent to give the desired solution at concentrations of 5 and 10 μM.

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Procedures on the detection of ethylene released during fruit ripening. Passion fruits (346

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g) were loaded in a capped gas-tight jar (2.25 L), and then the outlet was closed. After incubating

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the jar with a given time (2, 4, 6, and 8 h), sample gas (2.0 mL) was taken from the headspace of

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the jar using a gas-tight syringe with a long needle. The sample gas was bubbled slowly to the

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CH2Cl2 solution (2.0 mL) in a capped cuvette to dissolve the ethylene. To this solution, the probe

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stock solution (10 µL) was added and the mixture was shaken for 3 min. The fluorescence intensity

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of the solution was recorded from the fluorescence spectra in the range 380-600 nm using a 365

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nm excitation wavelength and a 500 nm/min scan rate. The ethylene released from the apple (366

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g), banana (360 g), and avocados (375 g) were determined using the same procedure as that of the

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passion fruit.

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The concentration of ethylene in the jar (Cppm) was measured using the following equation: I 𝐶𝑝𝑝𝑚 = ( ― 1.042)/0.044 𝐼0 The ethylene releasing rate of these fruits was calculated using the following equation: S=

∆𝑝𝑝𝑚 × 𝑉𝑗𝑎𝑟 𝑊𝑓𝑟𝑢𝑖𝑡 × ∆ℎ 8

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Where S represents speed rate of ethylene release, Δppm represents the ethylene concentration

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change in the jar, Vjar represents the volume of the jar, Wfruit represents the weight of selected fruit,

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Δh represents the incubation time of selected fruit.

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Statistical Analysis: One-way analysis of variance (ANOVA) and Tukey’s test using SPSS 17.0

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software for Windows (SPSS Inc., Chicago, IL) were applied to evaluate the significant differences

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among different fermentations (P < 0.05).

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RESULTS AND DISCUSSION

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Design and synthesis of the fluorophore-tagged Grubbs catalysts-based probes. We

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designed and prepared three fluorophore-tagged Grubbs catalysts that were responsive to ethylene

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sensitively and their application for sensitive detection of ethylene released during the fruit

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ripening process. Our design strategy to the fluorophore-tagged Grubbs catalysts based probes

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were illustrated in Figure 1. To obtain the ideal probe with optimal sensitivity, the fluorophores

178

with high fluorescence quantum yields were chosen and attached to the Grubbs catalyst through

179

the vinyl group by metathesis reaction. Moreover, the fluorescence of the resulted complex should

180

be weak if there were energy or electron transfer between fluorophores and Ru center.

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Taking these factors into consideration, we first prepared compound 2 (Figure 1), a BODIPY

182

tagged Ru complex via the metathesis reaction between the first generation Grubbs catalyst and

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the fluorophore 1. The design and preparation of these probes were reasonable and easy, with high

184

yield. The maximal absorbance and emission wavelengths of probe 2 were centered at 528 and

185

545 nm, respectively. The probe itself showed relatively weak fluorescence suggesting the

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effective fluorescence quench by energy transfer between the fluorophore and Ru center. However,

187

when the dichloromethane solution of probe 2 was irradiated at 500 nm for 20 min, the

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fluorescence steadily increased over time, indicating that probe 2 was unstable under the assay 9 ACS Paragon Plus Environment


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conditions that may be needed for ethylene monitoring. Furthermore, 1H NMR spectra of

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compound 2 showed that it was oxidized over time to fluorescent BODIPY-CHO. We speculate

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that BODIPY group coupled directly to the Ru metal center may act as a photosensitizer30, 31 which

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may induce the decomposition of the probe under irradiation.

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In the hope to reduce the photosensitivity and increase the stability of the fluorophore-tagged

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Grubbs catalyst, we prepared probe 4, in which the BODIPY group is separated from the Ru center

195

through pyrazine ring. Dichloromethane solution of compound 4 showed weak fluorescence likely

196

due to efficient quench effect of the FRET mechanism. Moreover, upon treatment of probe 4 with

197

ethylene gas, 8-fold fluorescence enhancement accompanied by bright green fluorescence color

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was obtained, suggesting a potential detection system for ethylene. However, we found that

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compound 4 was somewhat unstable after 20 min irradiation. Therefore, although probe 4

200

exhibited increased stability compared with compound 2, it is still not an ideal fluorescent probe

201

for ethylene analysis in assay conditions.

202

We suspected that, although BODIPY has high quantum yields, its property as photosensitizer

203

could compromise the stability of the probes. We then turned to pyrene, which has high

204

fluorescence quantum yield but is a poor photosensitizer. The pyrene-based probe 6 was

205

synthesized readily in high yield by attaching the pyrene group to Ru center by metathesis reaction

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between the first generation Grubbs catalyst with an excess amount of 1-vinylpyrene, 5, which

207

showed the fluorescence maximum at 394 nm and a shoulder peak at 414 nm exhibiting high bluish

208

violet fluorescence. The dichloromethane solution of 6 showed weak aquamarine fluorescence

209

color with maximum emission at 468 nm and a shoulder peak at 500 nm, indicating a

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bathochromic-shift effect when compound 5 was bound to Ru metal center. This result was in

211

agreement with those observed with pyrene functionalized Ru nanoparticles having Ru carbene π 10 ACS Paragon Plus Environment

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bonds, suggesting an extended conjugation of metal-carbene π bond which may lead to the

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appearance of new emission peaks in the lower energy region, allowing the pyrene moieties to

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behave analogously to their dimeric counterparts32. Attempts to synthesize the second generation

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Grubbs catalyst based probe using similar procedure failed while only the coupling product of 1-

216

vinylpyrene was observed. Somehow the second generation Grubbs catalyst acted as a metathesis

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reaction catalyst was likely due to its higher catalytic activity comparing to the first generation

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one. UV/Vis absorption spectra of the reaction mixture showed that absorption peak at 441 nm for

219

the probe 6 had disappeared and new absorption bands emerged at 495 nm, 360 nm, and 285 nm,

220

which can be attributed to the reaction product and the released of compound 5. Consistently, the

221

1H

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the probe solution was bubbled with 500 ppm ethylene for one min, the intensity of the signals

223

belonging to the probe disappeared gradually, while several new peaks appeared which can be

224

assigned to compound 7 and compound 5. The

225

probe 6 to compound 7 (Figure S7). These results suggested that 6 reacted with ethylene via alkene

226

metathesis to give compound 7 and the highly fluorescent species 1-vinylpyrene in quantitative

227

yield (Figure 2B).

NMR spectra of the reaction mixture showed that compound 5 was released (Figure 2A). When

31P

NMR also shows the transformation of the

228

When the probe 6 was treated with ethylene gas at an ambient temperature in dichloromethane,

229

the fluorescence intensity of the solution was turned on significantly and reached its maximum

230

within three minutes. Without ethylene, no significant fluorescence enhancement was observed

231

when probe 6 was irradiated with excitation light at 365 nm for 25 min, indicating relatively good

232

photostability. In addition, compound 6 in the solid state was not sensitive to air or humidity and

233

could be kept in the refrigerator at 4 °C for 3 months without any decomposition. In brief, among



234

the three synthesized probes, compound 6 showed the best sensing performance with high signal-

235

to-noise ratio and good stability (Figure 3).

236

Fluorescence responses of the probe toward ethylene. To test the sensitivity, an ethylene stock

237

solution was prepared by bubbling ethylene gas to CD2Cl2 to give the NMR spectrum of the

238

resulting solution. The ethylene concentration was calculated from the integration of the ethylene

239

peak and compared with the added internal standard (methyltriethoxysilane). Upon addition of an

240

increased amount of ethylene, the emission intensity of probe 6 (5.0 μM) that was dissolved in

241

CH2Cl2 at 468 nm decreased gradually, whereas the emission at 394 nm increased significantly,

242

resulting in fluorescence enhancement (Figure 4A) by ca 20-fold when the concentration of

243

ethylene reached 20 μM. Moreover, the fluorescence enhancement has a good dose-response

244

relationship (R2 = 0.992) with the concentration of ethylene in a wide concentration range (Figure

245

4A inset). In addition, this fluorescence enhancement could be easily visualized with the probe

246

solution changing in color from green to intense bluish violet under a 365 nm UV lamp (Figure

247

4A inset image).

248

For detection of gaseous ethylene, various concentrations of ethylene gas in the air were first

249

prepared and added to the solutions of 6 in dichloromethane with a gas-tight syringe. The

250

fluorescence intensity of the solution was measured after three minutes. A good ethylene dose

251

response was observed for the fluorescence intensity at 394 nm, with linear dependence in a wide

252

range of 0-200 ppm (R2 = 0.990), which could be used for the quantification of ethylene gas (Figure

253

4B). The limit of detection (LOD) for ethylene was determined at 0.9 ± 0.016 ppm in the air, which

254

is sufficiently sensitive to test the ethylene gas concentrations released by the fruits. Although the

255

LOD is much lower than that of GC, which has been developed for decades, it is sufficiently

256

sensitive to test the ethylene gas concentrations released by the fruits. The probe also has other 12 ACS Paragon Plus Environment

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advantages including low cost, mobility, visual detection, and real-time detection. Our probe has

258

the best sensitivity among the developed fluorescent probes reported up till now. Our work opened

259

up a new avenue of application of fluorescent probes for bioanalytical chemistry of ethylene. We

260

believe more fluorescent probes with comparable sensitivity to GC can be developed.

261

Selectivity. The probe 6 not only exhibited good sensitivity for the detection of ethylene but also

262

showed good selectivity (Figure 5A). Other possible coexisting species such as acetonitrile,

263

ethanol, ethyl acetate, water, toluene, THF, 1-propanol, and 1-butanol could not turn on the

264

fluorescence at the same concentration as ethylene, which gave a remarkable fluorescence

265

enhancement. In addition, the response of the probe to other gaseous species including CO2, NO2,

266

SO2, NH3, and H2S were also examined under the same conditions as ethylene (50 ppm). CO2,

267

SO2, and H2S only enhanced the fluorescence intensity slightly, while NO2 and NH3 only induced

268

slight fluorescence quench, since these species may induce the decomposition of Grubbs catalyst

269

slowly.27 It is worth mentioning that no apparent interference was obtained in fluorescence

270

intensity of solution 6 in the presence of other potential coexisting species at the concentration of

271

50 ppm (Figure 5B). The good selectivity could be attributed to the high tolerance of Grubbs

272

catalyst for other functional group and reaction priority to vinyl group.

273

Fluorescence determination of ethylene released from ripening fruits. Climacteric fruits

274

increase respiration and ethylene biosynthesis rates during fruit ripening. Ethylene formation

275

during fruits ripening has been used as a maturity index to determine the time of harvest. The

276

control of ethylene concentration is also of great importance for the ripening and storage of fruits.

277

Thus, it would be very useful to be able to monitor the ethylene release conveniently in ambient

278

air around the fruits. To demonstrate the utility of 6 in such application, we developed a simple

279

method for determining the ethylene released during fruits ripening process. As shown in Figure 13 ACS Paragon Plus Environment


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6A, weighed fruits were enclosed in an airtight jar. At a given time, 2 mL of gas sample was taken

281

from the headspace of the jar by an air-tight syringe and bubbled slowly into the dichloromethane

282

solvent in a cuvette with a rubber cap. Probe 6 solution was then added to the same cuvette

283

immediately. Then the fluorescence intensity of the solution was measured after three minutes.

284

The ethylene releasing dynamics of the different fruits were determined by monitoring the ethylene

285

concentrations over time. The response curves of the different fruits were shown in Figure 6B. For

286

the four fruits measured, there was a linear increase of ethylene concentrations with increased

287

storage time. Passionfruit gave the most remarkable response followed by avocado, banana, and

288

apple. From the slopes of the curves, the rates of ethylene released for different fruits were

289

determined in a range of 5-80 μL/kg/h (Figure 6C). The result was consistent with previous

290

reports.8

291

In summary, we have demonstrated that the fluorophore-tagged first generation Grubbs catalysts

292

are promising for rapid and sensitive visual detection of ethylene as well as quantitation of ethylene

293

released during fruits ripening. In particular, probe 6 showed the best ethylene sensing

294

performance with good stability. Our finding would open up a new avenue of application of Grubbs

295

catalysts for bioanalytical chemistry of alkenes that are of critical importance for plant science.

296

ASSOCIATED CONTENT

297

Supporting Information

298

Synthesis of the three probes 2, 4, and 6, decomposition route of compound 2 under light, the

299

reaction of 1-vinylpyrene with 2nd generation Grubbs catalyst, ANOVA data regarding selectivity

300

of probe 6, characterization of the synthesized probes 2, 4, and 6, fluorescence and absorption


Page 14 of 26

Page 15 of 26


301

responses of probe 6 toward ethylene, the images of weighted fruits which are enclosed in the gas-

302

tight jar. This material is available free of charge via the Internet at http://pubs.acs.org.

303

FUNDING SOURCES

304

The work was supported by Singapore Ministry of Education for financial support (grant no:

305

MOE2014-T2-1-134), the National Natural Science Foundation of China (grant no: 21475134,

306

21775042, 2160151), and Natural Science Foundation of Jiangsu, China (grant no: BK20141219).

307

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389 390 391 392

FIGURE CAPTIONS

393

Figure 2. The 1H NMR spectra of A) probe 6; (B) probe 6 bubbled with 500 ppm ethylene; (C) probe 6

394

bubbled with excess ethylene. (D) Mechanism of ethylene sensing using pyrene tagged Grubbs catalyst

395

probe 6.

396

Figure 3. Normalized fluorescence enhancement of the three probes, 2, 4, and 6, treated with excess

397

ethylene gas and control. All data were taken 20 min after treatment.

398

Figure 4. (A) The emission spectra of the probe 6 (5 μM) exposed to different concentrations of ethylene

399

(0-30 μM). Inset shows the linearity relationship of I/I0 as a function of the concentration of ethylene,

400

where I0 and I represent the fluorescence intensity at 394 nm (λex = 365 nm) before and after the addition

401

of ethylene 3 min, respectively. (B) Linearity relationship of fluorescence enhancement as a function of

402

the ethylene gas concentration in the range of 0-200 ppm.

403

Figure 5. (A) Selectivity of the probe 6 (5 μM) for ethylene, determined as the fluorescence response

404

after addition of these species at 5 and 10 μM. (B) Selectivity and interference of the probe 6 (5 μM) for

405

ethylene in the presence of other gaseous species at 50 ppm. I0 and I represent the fluorescence intensity

406

at 394 nm (λex = 365 nm) before and after the addition of ethylene, respectively.

407

Figure 6. (A) Procedures for determining the ethylene released from ripening fruits using probe 6. (B)

408

Fluorescence intensity changes of the probe exposed to ethylene released from different fruits at different

409

incubation time (2-8 h). (C) Ethylene releasing rates of different fruits.

Figure 1. Illustration of fluorescence turn-on detection of ethylene and preparation of the fluorophore-Ru based probes.

410



1st generation Grubbs catalyst

FL

Page 20 of 26

PCy3

FL

Cl

C2H4

Ru

ET

Cl

FL

H

Metathesis reaction

PCy3

FL = Fluorophore

H

Highly Fluorescent

Weakly Fluorescent

N N N F

B N F

N B N F F

3

1 Grubbs catalyst 1st PCy3

5 Grubbs catalyst 1st

Grubbs catalyst 1st PCy3

PCy3

Cl

Cl Ru

Ru

Cl

Cl PCy3

Cl

PCy3

N

PCy3

N

N B N F F

N B N F F

4

2

Figure 1


Cl

Ru

6

Page 21 of 26


Figure 2



Figure 3


Page 22 of 26

Page 23 of 26


Figure 4



Figure 5


Page 24 of 26

Page 25 of 26


Figure 6



Table of Contents Graphic


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Rapid and Visual Detection and Quantitation of Ethylene Released

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