Synthesis of Copper-Chelates Derived from Amino Acids and Evaluation of Their Efficacy as Copper Source and Growth Stimulator for Lactuca sativa in Nutrient Solution Culture

Article abstract of DOI:10.1021/acs.jafc.7b03809

Five tetradentate ligands were synthesized from l-amino acids and utilized for the synthesis of Cu(II)-chelates 1-5. The efficacy of Cu(II)-chelates as copper (Cu) source and growth stimulator in hydroponic cultivation was evaluated with Lactuca sativa. T

Full text of DOI:10.1021/acs.jafc.7b03809

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Article
Synthesis of Copper-Chelates Derived from Amino Acids and
Evaluation of Their Efficacy as Copper Source and Growth
Stimulator for Lactuca Sativa in Nutrient Solution Culture
Narongpol Kaewchangwat, Sattawat Dueansawang, Gamolwan Tumcharern, and Khomson Suttisintong
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03809 • Publication Date (Web): 26 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
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Synthesis of Copper-Chelates Derived from Amino Acids and Evaluation of
Their Efficacy as Copper Source and Growth Stimulator for Lactuca Sativa
in Nutrient Solution Culture
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Narongpol Kaewchangwat, Sattawat Dueansawang, Gamolwan Tumcharern
and Khomson Suttisintong*
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National Nanotechnology Center (NANOTEC), National Science and Technology
Development Agency (NSTDA), 111 Thailand Science Park, Thanon Phahonyothin,
Tumbon Khlong Nueng, Amphoe Khlong Luang, Pathum Thani, 12120, Thailand
khomson@nanotec.or.th
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Abstract
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Five tetradentate ligands were synthesized from Lꢀamino acids and utilized for the synthesis
of Cu(II)-chelates 1ꢀ5. The efficacy of Cu(II)ꢀchelates as copper (Cu) source and growth
stimulator in hydroponic cultivation was evaluated with Lactuca sativa. Their stability test
was performed at pH 4–10. The results suggested that Cu(II)-chelate 3 is the most pH
tolerant complex. Levels of Cu, Zn and Fe accumulated in plants supplied with Cu(II)ꢀ
chelates were compared with those supplied with CuSO4 at the same Cu concentration of 8.0
ꢀM. The results showed that Cu(II)-chelate 3 significantly enhanced Cu, Zn and Fe content
in shoot by 35%, 15% and 48%, respectively. Application of Cu(II)-chelate 3 also improved
plant fresh and dry matter yield by 54%. According to the results, Cu(II)-chelate 3
demonstrated the highest stimulating effect on plant growth and plant mineral accumulation
so that it can be used as an alternative to CuSO4 for supplying Cu in nutrient solutions and
enhancing the plant growth.
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Keywords: nutrient solution, copper chelate, amino acid derived chelator, plant copper
uptake, hydroponics
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Introduction
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Copper (Cu) is one of essential micronutrients that play a significant role in a number of
plant physiological processes such as photosynthesis and respiratory electron transport
chains.1, 2 It also acts as a cofactor or a part of the prosthetic group of many key enzymes
involved in different metabolic pathways, including ATP synthesis.3 When copper is not
available, plants develop specific deficiency symptoms, most of which affect young leaves
and reproductive organs.4 Thus, copper deficiency can alter essential functions in plant
metabolism resulting in low crop productivity. Recently, polymerꢀencapsulated copper
nanoparticles have been applied to solve copper deficiency symptoms in plants,5 and to
improve plant crop productivity. Moreover, encapsulation of copper cations and Trichoderma
viride in chitosan/alginate microcapsule were used as biological and chemical agents in plant
nutrition and protection.6
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Higher plants accumulate copper from soil solution mainly as Cu(II). In nutrient solution
cultures, Cu ion is prepared at a desirable concentration (0.5ꢀ12.5 ꢀM)7ꢀ12 of this element for
the plant.13 The most common Cu source used in nutrient solutions is Cu(II) ion from CuSO4.
When CuSO4 is added to a nutrient solution which contains an abundance of anions such as
3ꢀ
phosphate (PO4 ) or hydroxide (OHꢀ), its precipitation can occur.14ꢀ17 The precipitation
causes a lower concentration of free Cu ion in nutrient solutions and results in insufficient
amount of Cu for plant uptake. This contributes to plant disorder.18ꢀ22
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To maintain sufficient amount of certain micronutrients (e.g. Fe, Zn, Mn, Cu) in nutrient
solutions, free ligands or chelators are required to form a stable and soluble complex (chelate)
of micronutrients.23ꢀ33 This also helps translocation of micronutrient to most parts of the
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plants.34ꢀ37 To date, many synthetic chelators such as EDTA,23 EDDHA,24ꢀ27 IDHA,28ꢀ29 or
DTPA30 have been used commercially in hydroponic cultivation or spray nutrient solution
especially for iron (Fe)23ꢀ30 and manganese (Mn)31, 32. Application of Cuꢀchelates such as Cuꢀ
EDTA and CuꢀDTPA in soil culture demonstrates a significant improvement of nutritional
value to the crop.38 However, their application in hydroponic cultivation is limited.
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Amino acids are cheap and abundant sources for chelates and have been widely used as
agrochemical products. It has been shown that uses of some amino acids in nutrient solutions
improves micronutrient uptake by crops.39 Amino acids have the ability to coordinate metal
ions via their carboxyl and amino groups.40, 41 They are less sensitive to photodegradation or
their degradation is completely biological.42 There are some results that indicate negligible
degradation of amino acids in nutrient solutions,43 however the degradability of metalꢀamino
acid complexes is still less than free amino acids.44, 45 In addition, amino acids are sources of
nitrogen for plant nutrition46 and most plants can directly absorb amino acids and use them in
their physiological structures and processes.43, 47, 48 This may result in less accumulation of
free ligand in the media and help maintain other micronutrient balance.
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Although a free amino acid (bidentate ligand) can be a good chelator, it may form an
unstable metalꢀchelate complex in a hydroponic nutrient solution which consists of high
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concentration of anions such as OHꢀ, PO43ꢀ or HPO4 . The first objective of this research is,
therefore to synthesize tetradentate ligands from amino acids and evaluate their efficiency as
copper chelators. To achieve this goal, four Lꢀenantiomers (natural form in plants) of amino
acids including Lꢀphenylalanine (Phe), Lꢀleucine (Leu), Lꢀisoleucine (Ile) and Lꢀvaline (Val)
were chosen as starting materials to synthesize Cu chelators. These amino acids were selected
because of their significance in plant and human nutrition. It has been shown that systemic
application of Lꢀphenylalanine increases plant resistance to vertebrate herbivory because this
significantly increases the phenolic pool of oilseed rape.49 Additionally, Lꢀphenylalanine is a
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precursor for many substance including ubiquinone which is an essential respiratory
cofactor.50 Valine, leucine and isoleucine together form a unique group of soꢀcalled
branchedꢀchain amino acids (BCAAs) needed for human nutrition. These essential amino
acids are critical for protein synthesis and normal plant growth, while also providing
precursors for a number of secondary metabolites such as; cyanogenic glycosides,51
glucosinolates,52 and acylꢀsugars.53 In addition, valine has been reported to act as a metabolite
for the respiration of higher plants and yield CO2.54 Our next objectives are to form Cu(II)ꢀ
chelates (from tetradentate ligands), characterize and confirm their structures, investigate
their stability under various pH conditions, and evaluate their efficacy against Lactuca sativa
L., a red oak leaf lettuce commercially grown hydroponically in Thailand. To the best of our
knowledge, there are only a few studies showing the effect of Cu(II)ꢀchelates on plant growth
in hydroponic cultivation.12 Thus, the investigation of stimulating effects of Cu(II)ꢀchelates
on plant growth and mineral accumulation is our ultimate target.
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Materials and Methods
Chemicals
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Lꢀphenylalanine, Lꢀleucine, Lꢀisoleucine and Lꢀvaline were purchased from Acros
Organics. Ethylene glycol, ethylenediaminetetraacetic acid and pꢀtoluenesulfonic acid were
purchased from Aldrich Chemical Co. Pyrocatechol was purchased from Tokyo Chemicals.
Copper sulfate pentahydrate (CuSO4·5H2O), calcium nitrate (Ca(NO3)2), iron sulfate
heptahydrate (FeSO4·7H2O), potassium nitrate (KNO3), magnesium sulfate (MgSO4),
ammonium dihydrogen phosphate (NH4H2PO4), boric acid (H3BO3), manganese sulfate
(MnSO4), zinc sulfate (ZnSO4), ammonium heptamolybdate ((NH4)6Mo7O24), sodium
hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Carlo Erba. Acetone
and toluene were purchased from Fisher Scientific. Seeds of red oak leaf lettuce (Lactuca
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sativa L.) were purchased from Chai Tai Co., Ltd. All reagents received were analytical grade
and used as received unless stated otherwise. Deionized water (DI water) was used in
experiments. All stock solutions were kept in the refrigerator at 4 °C prior to use at room
temperature.
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Instrumentation
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Fourier Transform Infrared (FTꢀIR) spectra were recorded using a Thermo Scientific
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Nicolet 6700 spectrometer in ATR mode. H NMR (500 MHz) spectra and C NMR (125
MHz) with complete proton decoupling spectra were recorded by a Bruker AVꢀ500 in
Fourier transform mode. Spectra were obtained in D2O solutions and chemical shifts in ppm
(part per million) are quoted relative to the residual signals of D2O (δH 4.79 ppm). Data are
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reported as follows: chemical shifts, multiplicity, coupling constant. Multiplicities in the H
NMR spectra are described as: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad; coupling constants (J) are reported in Hz. High resolution mass spectra (HRMS)
were recorded using Bruker micrOTOF mass spectrometer (LCꢀESIꢀTOF) and reported with
ion mass/charge (m/z) ratios.
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Methods
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General Procedure for the Synthesis of Amino Acid-Derived Chelators
To a stirred suspension of 8.00 mmol Lꢀamino acid (Lꢀphenylalanine, Lꢀleucine, Lꢀ
isoleucine or Lꢀvaline) and 4.00 mmol ethylene glycol (or pyrocatechol) in 15 mL toluene
was added 8.00 mmol pꢀtoluenesulfonic acid and the resulting mixture was heated to 115 °C
with stirring for overnight. The reaction mixture was cooled to room temperature and 20 mL
acetone was added. The precipitate was filtered, washed with acetone and dried under
reduced pressure to give the corresponding chelator which was used in the next step without
purification.
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General Procedure for the Synthesis of Cu(II)-Chelate Complexes
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Cu(II)ꢀchelate complexes (Cu(II)-chelate 1-5) were prepared using amino acidꢀderived
chelators 1-5 as complexing agents. (Their characterization is available in the Supporting
Information, SI). A solution of 1.25 mmol chelator 1 in 100 mL DI water was slowly added
to a solution of 1.25 mmol CuSO4 in 100 mL DI water. The mixture was heated to 45°C and
stirred for 2 h. The mixture was cooled to room temperature and water was removed by
freezeꢀdrying technique to yield blue solid of Cu(II)ꢀchelates. The products were washed with
cold ethanol followed by diethyl ether and airꢀdried. All complexes were characterized by
NMR and FTꢀIR.
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Hydroponics Cultivation
Seeds of red oak leaf lettuce (Lactuca sativa L.), most commonly grown hydroponically in
Thailand, were thoroughly rinsed with DI water and germinated on moist sponge for
hydroponic cultivation in a halfꢀfilled water tray which was placed in incubator at 28 °C.
After 7–10 days, uniformꢀsize seedlings which had two first leaves (primary leaves) were
transferred to PVC lids that fit tightly over 1ꢀL glass bottle containers which were placed in a
greenhouse under controlled conditions, with a 12ꢀh light period and 32/29 °C day/night
temperature, and 65ꢀ75% relative humidity. The bottles were wrapped with black
polyethylene to prevent light from reaching the roots and nutrient solution. One plant was
grown in each bottle. A basic nutrient solution was prepared in DI water (electrical resistivity
= 18 Mꢁ cmꢀ1). The basic nutrient solution contained 4.0 mM Ca(NO3)2, 0.055 mM Feꢀ
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EDTA, 8.0 mM KNO3, 2.0 mM MgSO4, 1.3 mM NH4H2PO4, 0.046 mM H3BO3, 9.5 ꢀM
MnSO4, 0.77 ꢀM ZnSO4, 20.2 nM (NH4)6Mo7O24 adjusted to pH 6.0 with NaOH or HCl as a
buffer. Copper was supplied from six different sources: CuSO4 and Cu(II)-chelate 1-5 and
the Cu concentration in all treatments was 8.0 ꢀM (2 ppm). Plants were harvested
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approximately 4 weeks after seeding and divided into shoot and root. Fresh and dry matter
yields of shoot and root in each bottle were determined.
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Remaining Concentration (ppm) of Copper in Nutrient Solutions
The remaining concentration of Cu (in ppm) in the CuSO4 solution and the Cu(II)-chelate
3 solution were investigated to confirm the absorption of Cu into the plants. 5.0 mL of
nutrient solutions were sampling at day 4, 7, 14, 21, and 28 after the seedlings were planted
in the nutrient solutions. At day 18, 1 ppm of CuSO4 or Cu(II)-chelate 3 was added to each
bottle. The calibration curve was prepared using standard CuSO4 solutions at 0.05, 0.25, 0.75,
1.25 and 2.50 ppm. Analyses of Cu contents were carried out with an atomic absorption
spectrometer (AAS) model PinAAcle 900F, PerkinꢀElmer.
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Elemental Analyses of Cu, Zn and Fe content in Dry Plant Matter
Plant materials were dried in a vacuumꢀdried oven at 50 °C to a constant weight and
ground to a fine powder form in a Milestone ETHOS labstation. Dry samples (0.10 g) were
placed in TFM vessels. 65% HNO3 (14 mL) and 30% H2O2 were added dropwise to the
vessels, then the vessels were gently swirled to homogenize the samples. The vessels were
put under microwave digestion at 1000 watt, 200 °C for 10 minutes. The final solution was
diluted to meet the range requirements of the analytical procedures. Analyses of Cu, Zn and
Fe contents in shoots and roots were carried out with an inductively coupled plasma atomic
emission spectrometer (ICPꢀAES) model Optima 7300DV, Perkin Elmer.
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Statistical Analysis
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Analysis of variance (using oneꢀway ANOVA) and Tukey test were applied to the data of
sample set at 95% confidential interval (p < 0.05). Statistical processing was carried out using
Minitab Release 17.2.1 software (Minitab Inc., State College, PA, USA).
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Results and Discussion
Synthesis of Chelators 1-5
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The amino acidꢀderived chelators 1ꢀ5 were successfully synthesized as outlined in
Scheme 1 and 2. Chelator 1, 3, 4 and 5 were designed based on different amino acids to
investigate the effect of amino acid types on chelation efficacy. Chelator 2 was synthesized to
compare the chelation efficacy when bridging moiety between two amino acid units is
changed from diethylene glycol (Scheme 1) to pyrocatechol (Scheme 2). All products were
white solid with the reaction yields of 23ꢀ88%. The difference in reaction yields may result
from different solubility of amino acid starting materials in the reaction mixtures. All
chelators were characterized using FTꢀIR, NMR and HRMS to confirm the structures. The
FTIR spectra of chelator 1, 3, 4 and 5 showed an absorption band in the range of 1735ꢀ1750
cmꢀ1 indicating the stretching vibration of the newly formed ester carbonyl group (C=O).
Moreover, the strong absorption band at 1100ꢀ1200 cmꢀ1 derived from the stretching mode of
the CꢀO bond also confirmed the presence of an ester moiety. The bands in these regions are
distinguished from simple amino acid starting materials and this ensures the ethylene linkage
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on the chelator molecules. In addition, the H NMR spectra of chelators 1, 3, 4 and 5
exhibited the signals of the ethylene linkage moiety at around
δ 3.80ꢀ4.20 ppm with the
integration of four protons. This ester formation was also confirmed by 13CꢀNMR and
DEPT135 experiments which show extra ethylene CH2ꢀcarbons in the
δ 60ꢀ70 ppm region.
The FTIR spectra of chelator 2 showed an absorption band of the ester carbonyl (C=O)
stretching at 1747 cmꢀ1 and ester CꢀO stretching at 1122 cmꢀ1. Besides, the 1Hꢀ and 13CꢀNMR
spectra of chelator 2 displayed the proton signals at
δ 7.05ꢀ7.15 (4H) and carbon signals at δ
125ꢀ130 ppm. These signals indicate the presence of pyrocatechol aromatic moiety. The high
resolution mass spectra of all chelators were relevant to the molecular weight of the
structures.
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ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Scheme 1 Synthesis of chelator 1, 3-5
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ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Scheme 2 Synthesis of chelator 2
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
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Characteristics of Cu(II)-chelate 1-5 Complexes
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Infrared Spectroscopy
The FTIR spectra of the Cu(II)-chelate 1-5 complexes showed an absorption in the 4,000ꢀ
500 cmꢀ1 region similar to the chelators, which generally act as tetradentate ligands.
Predominant vibrations for the complexes are associated with ν(C=O), ν(CꢀO), ν(NH2). The
observed vibrational bands for –NH2 groups appear around 3,100ꢀ3,500 cmꢀ1 as very broad
signals due to the effect of intermolecular interaction in the solid state. The carbonyl group
(C=O) of the chelates has approximately the same frequency to the chelators at around 1,590
– 1,690 cmꢀ1.
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1H- and 13C-NMR tritration of CuSO4 and Chelator 4
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Fig. 1 Structure of chelator 4. a) Chelator 4 with numbering system; b) Proposed structure of
Cu(II)-chelate 4 complex
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
The formation of Cu(II)ꢀchelates in aqueous solution was demonstrated using chelator 4 as
a model study and the experiments were performed using 1Hꢀ and 13CꢀNMR titration in D2O.
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Chelator 4 was selected for this experiment because its H NMR and C NMR signals are
uncomplicated with enough structural detail. The results revealed that the suitable ratio of
Cu(II) to chelator 4 to form the chelate complex is 1:1. The proposed structure of Cu(II)-
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chelate 4 complex was also shown in Fig. 1 based on the NMR experiments. The NMR
method to confirm the geometry of the chelate complex has been studied and the report
1
suggested that Cu(II) tended to form a square planar geometry.55 In our study, the HꢀNMR
spectra of chelator 4 show signals of protons at C1 and C1’ position (Fig. 1a) at 3.81 and
4.31 ppm as triplets (Fig. 2). These triplet signals are eventually broader after the ratio of Cu
increased to 1:1 ratio. The multiplet signals at around 4.00 ppm (1H) and 4.15 ppm (1H)
indicate the differentiation of protons at C3 and C3’. When the ratio of Cu increased to 1:1
ratio, these proton signals collapsed to a new broad peak at 4.13 with the integration of 2H,
revealing a lack of distinction of C3ꢀH and C3’ꢀH after the formation of Cu(II)-chelate 4
complex. This can be illustrated by structure of Cu(II)-chelate 4 (Fig. 1b) in which a C2
symmetry is present in the molecule after the complex was formed. The protons at C3 and
C3’ or so called Hc were in the C2 environment resulting in the collapsing of the initial
signals to a broad peak at 4.13 (2H).
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Fig. 2 1HꢀNMR spectra of Cu(II)-chelate 4 (δ 3.70 – 4.40 ppm)
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
13
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The CꢀNMR spectra of chelator 4 contain signals of C4 and C4’ at 38.7 and 38.9 ppm
(Fig. 3). After the ratio of Cu increased, these two carbon signals collapsed to a single peak at
38.7 ppm with doubled intensity. This can also be explained by the structure of Cu(II)-
chelate 4 (Fig. 1b) where the molecule has a C2 symmetry. The carbon signals of chelator 4
at 51.4 and 51.6 ppm show the distinction of carbon at C3 and C3’. These two signals
eventually collapsed to a peak at 51.4 ppm after the ratio of Cu increased to 1:1 ratio.
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The changes in chemical shifts of chelator 4 in H NMR and 13C NMR spectra after
addition of CuSO4 indicate the formation of Cu(II)-chelate 4 at 1:1 ratio of chelator 4 to
CuSO4.
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ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Fig. 3 13CꢀNMR spectra of Cu(II)-chelate 4 (δ 20.0 – 70.0 ppm)
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ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
pH Tolerance of Cu(II)-chelate 1-5 (Formation of Cu(OH)2)
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Since pHs of nutrient solutions significantly affect bioꢀavailability of Cu in hydroponic
nutrient solution, the stability of Cu(II)-chelate 1-5 was investigated by means of pH
tolerance test at pH 4 – 10 (data shown in the SI Fig. S2). CuSO4 solution started
precipitating at pH 5 (picture not shown) while Cu(II)-chelate 2 and Cu(II)-chelate 4
solutions began precipitating at pH 4. Cu(II)-chelate 1 precipitated in the pH ranging from 6
– 10 while Cu(II)-chelate 5 could tolerate basic condition up to pH 8. Cu(II)-chelate 3 gave
no precipitation under the whole pH range (4 – 10). The results obtained from this test
suggested that Cu(II)-chelate 3 and Cu(II)-chelate 5 were stable in pH ranging from 5 – 8
which is the pH range used for hydroponic cultivation. This stability may result from a strong
interaction of Cu(II) ion and chelator 3 or 5 that prevents the complexes from reacting with
hydroxide ion.
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Gravimetric analysis (Measurement of Cu(OH)2 precipitate)
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The stability of Cu(II)-chelate complexes were relevant to gravimetric analysis of
Cu(OH)2 where Cu(II)-chelate 3 and 5 gave only small amount of the precipitate from the
solution in the whole range of tested pH (pH 5 – 8) (Fig. 4). The results revealed that basicity
of the solution had a great impact on precipitation of Cu(OH)2. When pH increased, more
Cu(OH)2 precipitated out from each solution. The culprit was from high concentration of
hydroxide ion (OHꢀ) at higher pH reacting with the Cu(II) ion and give precipitation (eq. 1
and eq. 2). Cu(II)-chelate 1, Cu(II)-chelate 2 and Cu(II)-chelate 4 gave as much precipitate
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as CuSO4 at pH 8. Cu(II)-chelate 5 gave about 5 times less precipitate than CuSO4. Cu(II)-
chelate 3 gave superior result by generating a minimal amount of precipitation throughout
the whole range of tested pH. These results suggested that Cu(II)-chelate 3 and 5 are the best
candidates suitable for nutrient solution that can tolerate a changing in pH.
CuSO4 (aq) + chelator (aq)
Cu(II)ꢀchelate (aq) + H2SO4 (aq)
Cu(OH)2 (s) + Na2[chelate] (aq)
•
•
•
•
(eq. 1)
(eq. 2)
Cu(II)ꢀchelate (aq) + 2NaOH (aq)
•
•
•
•
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Fig. 4 Gravimetric analysis of Cu(OH)2 from Cu(II)ꢀchelate solutions compared with CuSO4
at various pH (*Asterisks above the bars indicate a significance of p<0.05, n = 3.)
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ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Energy-dispersive X-ray spectroscopy (EDS) (Spectra shown in the SI Fig. S3-S6)
The EDS spectra of the precipitates from Cu(II)-chelate 4 and CuSO4 solution at pH 10
were compared with Cu(II)-chelate 4 complex and the standard Cu(OH)2. Cu(II)-chelate 4
was selected as a model study for this experiment because it gave the most precipitate. The
precipitate from Cu(II)-chelate 4 and CuSO4 solution at pH 10 gave similar spectra to the
spectra of the standard Cu(OH)2. This indicated that Cu(OH)2 was formed from Cu(II)-
chelate 4 and CuSO4 solution under high concentration of hydroxide ion (pH 10). The
formation of Cu(OH)2 also occurred in Cu(II)-chelate 1 and 2 treatments. The EDS spectra
of Cu(II)-chelate 4 complex shows the existence of chelator 4 by the presence of a strong
emission of C peak while the spectra of the precipitate from pH 10 solution shows no sign of
chelator signals. In case of Cu(II)-chelate 3 and Cu(II)-chelate 5 solutions at pH 10, the
formation of Cu(OH)2 was negligible (a small amount of Cu(OH)2 was observed).
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Root and Shoot Length
The results obtained from the stability test (pH tolerance test and gravimetric analysis)
suggested that Cu(II)-chelate 3 is the most stable chelate complex under various pH
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conditions so that it was used as a model study in the comparison of root and shoot length.
Root and shoot length of Lactuca sativa grown in nutrient solutions at 8.0 ꢀM of Cu (CuSO4
and Cu(II)-chelate 3) were compared at day 38 of the cultivation. A slight difference in root
length between CuSO4 (control treatment) and Cu(II)-chelate 3 solution was observed in the
whole range of cultivation time (data shown in the SI Fig. S7). However, the shoot length of
Lactuca sativa grown in Cu(II)-chelate 3 nutrient solution at day 38 was about 40% longer
than that grown in CuSO4 treatment (Fig. 5). This result suggested that Cu(II)-chelate 3
helped increase bioavailability of Cu for plant and consequently, the shoot length of plants
grown in Cu(II)-chelate 3 treatment was significantly higher.
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ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
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Fig. 5 Shoot length of Lactuca sativa grown in nutrient solution containing Cu(II)-chelate 3
and CuSO4 (control) at different days of cultivation.
(*Asterisks above the bars indicate a significance of p<0.05, n = 15)
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
The Remaining Copper Content in Nutrient Solutions
The remaining Cu content in nutrient solutions used to grown Lactuca sativa was
investigated at day 4, 7, 14, 21 and 28 of cultivation. The nutrient solution containing 1 ppm
of Cu (CuSO4 or Cu(II)-chelate 3) was added on day 18 to maintain the appropriate mineral
contents. Fig. 6 shows the decrease of total Cu content in control and Cu(II)-chelate 3
treatment. The results revealed that Lactuca sativa grown in Cu(II)-chelate 3 absorbed more
Cu than that grown in CuSO4 for the whole period of cultivation. At day 4, Cu was slightly
absorbed in both treatments. At day 7, 50% of Cu was consumed by Lactuca sativa grown in
Cu(II)-chelate 3 but only 20% was consumed in the case of CuSO4 treatment. Plants grown
in both solutions consumed more than 50% of Cu after day 14. This was relevant to the plant
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growth where in the case of Cu(II)-chelate 3, plants had higher shoot length than that of the
CuSO4 treatment.
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Fig. 6 Remaining Cu content in nutrient solution of Cu(II)-chelate 3 and CuSO4 (control)
treatments at different days of cultivation.
(*Asterisks above the bars indicate a significance of p<0.05,n = 15)
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
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Fresh and Dry Matter Weight
Application of Cu(II)ꢀchelate as a Cu source had an effect on the plant growth. Besides
micronutrients, amino acids are required for plant growth and development. The uptake of
amino acids by plants56ꢀ58 and their effect on the yield and quality of crops have been
extensively studied.59ꢀ62 Some significant effects of amino acids on growth stimulation are
activation of sugar synthesis and increased mRNA transcription resulting in increasing
protein content.59, 61, 62 Since the amino acids can stimulate chlorophyll biosynthesis, they can
significantly improve the photosynthesis rate.63, 64 Moreover, several research groups have
reported the growthꢀstimulating effect of amino acids.64, 65 Some Cu(II)ꢀchelates used in this
study showed a stimulating effect on plant growth. This effect was considered from the
significant enhancement of shoot and root fresh (data shown in the SI Fig. S8) and dry matter
yield (Fig. 7). The effect was dependent on the type of amino acid starting materials used to
synthesize the chelators. Cu(II)-chelate 3 and 5 significantly enhanced fresh and dry matter
weight of root and shoot of Lactuca sativa compared to CuSO4 while Cu(II)-chelate 1, 2 and
4 had no significant effect on root and shoot matter yields of Lactuca sativa. The positive
effect of the Cu(II)-chelate 3 on root and shoot matter yields on Lactuca sativa was greater
than that of the Cu(II)-chelate 5 due to its greater solubility and better pH tolerance.
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
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Fig. 7 Root and shoot dry matter weights of Lactuca sativa grown in nutrient solution
containing Cu(II)-chelate 3 and CuSO4 (control).
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(*Asterisks above the bars indicate a significance of p<0.05, n = 15)
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Cu, Zn and Fe Contents in Root and Shoot
While application of Cu(II)-chelate 3 resulted in a significant increase of root and shoot
Cu, Zn and Fe content, Cu(II)-chelate 1, 2, 4 and 5 gave slightly higher mineral contents
compared to CuSO4. Plants grown in Cu(II)-chelate 3 nutrient solution accumulated 35%
higher Cu in their shoots compared to those grown in CuSO4 treatment (data shown in SI,
Fig. S9). The Cu(II)-chelate 3 treatment also enhanced shoot Zn and Fe by 15% and 48%,
respectively. Moreover, the content of Cu in roots of plants grown in Cu(II)-chelate 3 was
two times higher than those grown in CuSO4 (Fig. 8). While Zn content in roots of plants
grown in Cu(II)-chelate 3 was comparable to those grown in the CuSO4 treatment, the root
Fe content was significantly greater (64% higher). It seems that enhanced Cu and amino acids
uptakes in the form of Cu(II)-chelates play important roles in improvement of plant growth
as well as Fe and Zn uptakes into plants.
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Fig. 8 Root mineral content of Lactuca sativa grown in nutrient solution containing Cu(II)-
chelate 3 and CuSO4 (control).
(*Asterisks above the bars indicate a significance of p<0.05, n = 6)
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
The results obtained from hydroponic cultivation indicated that using Cu(II)ꢀchelates
derived from amino acids (especially, Cu(II)-chelate 3 and 5) in nutrient solutions could
supply sufficient amounts of Cu for plant uptake. The growthꢀstimulating effect of Cu(II)-
chelate 3 was the greatest. In contrast, Cu(II)-chelate 1, 2 and 4 had no significant effect on
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both plant growth and micronutrient uptakes of Lactuca sativa. This is probably because of a
lower stability of these chelates in nutrient solution leading to Cu precipitation and less Cu
absorption into the plants. The uptake rate of Cu(II)ꢀchelates is dependent on the plant
cultivar and amino acid characteristics.66ꢀ68 Therefore, lower plant uptake of Cu(II)-chelate
1, 2 and 4 compared with Cu(II)-chelate 3 and 5 in the present study may result from
different types of amino acids that lead to different solubility and chelate uptakes.
Improved Cu nutritional status of plants supplied with Cu(II)ꢀchelates compared with
CuSO4 supplied plants could also be related to improved nutritional nitrogen from amino
acids. The plant Cu content and dry matter weights in Table 1 suggested a role of synthesized
Cu(II)ꢀchelates in the translocation of Cu and amino acids from roots to shoots. Translocation
of amino acids in the plant is an important process.69 Amino acid translocation occurs in both
the phloem and xylem.70 This process also helps translocation of nutrient elements,
particularly immobile elements (for example, Cu, Fe and Zn) in the plant.71 In a trend similar
to that of Cu, Zn and Fe content in shoot and root were enhanced in the presence of chelates.
This may be a result from enhancement of Cu uptake, which increases the accumulation of
other minerals for plant nutritional processes. Therefore, the stimulating effect on the growth
rate was observed.
In conclusion, the results obtained from this study suggested that Cu(II)–chelate 3 and 5
are stable in nutrient solutions for hydroponic cultivation. The difference in stability of
Cu(II)ꢀchelates was a consequence from different types of amino acid starting materials.
Cu(II)-chelate 3 significantly improved Cu uptake in shoot of Lactuca sativa by 35% higher
than the CuSO4 treatment at the same molar amount of Cu (8.0 ꢀM). Moreover, plants grown
in Cu(II)-chelate 3 accumulated higher level of Zn and Fe in shoot by 15% and 48%,
respectively. These effects on micronutrient translocation and accumulation also improved
plant dry matter weights of the crop by 54%. Therefore, the experiments performed in this
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work supported that Cu(II)-chelate 3 has a stimulating effect on the plant growth. According
to the results, Cu(II)ꢀchelates can be used as an alternative to CuSO4 for supplying Cu in
nutrient solutions and can improve crop yield for plants grown hydroponically. Further
research in economic and environmental issues is our future study in order to confirm that
these Cu(II)ꢀchelates can be used commercially.
389
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393
Supporting Information
394
395
396
397
398
399
400
401
402
403
404
405
406
407
The Supporting Information is available free of charge on the ACS Publications website.
Methods for chelator synthesis and characterization, methods for Cu(II)ꢀchelates
characterization and evaluation, Furrier TransformꢀInfrared Spectroscopy (FTꢀIR)
spectra of Cu(II)-chelate 3, pH tolerance test of Cu(II)-chelate 1-5, EnergyꢀDispersive
Xꢀray Spectroscopy (EDS) spectra of Cu(II)-chelate 4 and Cu(OH)2, bar charts of root
length, fresh matter weights, shoot mineral content and figures of effect of Cu(II)-
chelate 3 on plant growth (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +662ꢀ564ꢀ7100. Eꢀmail: khomson@nanotec.or.th
ORCID
Khomson Suttisintong: 0000ꢀ0002ꢀ8797ꢀ6959
Notes
The authors declare no competing financial interest.
408
409
410
Acknowledgments
This research was financially supported by the Thailand Development and Promotion of
Science and Technology Talents Project (DPST Research Grant 007/2557).
411
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Table
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Table 1 Dry matter weight and mineral contents of plants grown in different treatments
Cu source
CuSO4
Parts Dry Weight
(g)
Cu
(ppm)
Zn
(ppm)
Fe
(ppm)
Shoot
1.30±0.16
0.09±0.02
0.25±0.10
0.09±0.01
0.27±0.09
0.09±0.02
0.34±0.09
0.12±0.02
0.77±0.11
0.10±0.02
0.45±0.11
0.11±0.02
0.56±0.10
0.65±0.10
1.03±0.10
0.65±0.10
0.98±0.10
0.67±0.09
0.99±0.22
0.74±0.13
1.12±0.24
0.70±0.11
1.01±0.21
0.70±0.13
1.07±0.15
0.96±0.16
3.72±0.77
0.99±0.18
3.73±0.74
1.05±0.16
4.39±0.86
1.42±0.23
6.10±1.14
1.10±0.17
5.01±0.93
1.14±0.17
5.44±1.06
Root
Shoot
Root
Shoot
Root
Shoot
Root
Shoot
Root
Shoot
Root
0.38±0.11
1.17±0.25
0.42±0.09
1.27±0.15
0.39±0.07
1.76±0.19
0.83±0.12
1.27±0.20
0.46±0.09
1.51±0.28
0.56±0.09
Cu(II)-chelate 1
Cu(II)-chelate 2
Cu(II)-chelate 3
Cu(II)-chelate 4
Cu(II)-chelate 5
611
612
613
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 34
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Schemes
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616
Scheme 1 Synthesis of chelator 1, 3-5
617
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
618
619
Scheme 2 Synthesis of chelator 2
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621
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
ACS Paragon Plus Environment

Page 29 of 34
Journal of Agricultural and Food Chemistry
29 | P a g e
622
Figures
2+
H2O
Hc
O
b
a
Ha
Hb
O
NH
NH
C2
1
1'
Cu
7
7'
5'
O
O
O
O
Hb
Ha
O
O
6'
6
5
3'
3
2
2'
Hc
4
4'
NH2
H2N
H2O
4
Cu(II)-chelate 4
623
624
625
Fig. 1 Structure of chelator 4. a) Chelator 4 with numbering system; b) Proposed structure of
Cu(II)-chelate 4 complex
626
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628
629
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631
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633
634
635
636
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Chelator 4:Cu 1:1
Chelator 4:Cu 1:0.75
Chelator 4:Cu 1:0.50
Chelator 4:Cu 1:0.25
Chelator 4:Cu 1:0
Fig. 2 1HꢀNMR spectra of Cu(II)-chelate 4 (δ 3.70 – 4.40 ppm)
637
638
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
ACS Paragon Plus Environment