1. Introduction
In recent years, pesticides with phloem mobility [
1,
2] have received considerable attention due to their effective control of vascular pathogens [
3] and improved targeting and utilization efficiency, reducing their usage and associated environmental pollution [
4]. Since most pesticides do not have phloem mobility, it is necessary to develop strategies to guide the molecular design of pesticides and improve phloem mobility.
A mathematical model to associate phloem mobility with xenobiotic-physicochemical properties (acid dissociation constant and octanol-water partition coefficients, Log Kow and pKa) was established by Kleier et al. [
5]. Xenobiotics with a pKa between −0.5~4 and a Log Kow between 3~6 may have phloem mobility. In previous reports, the Kleier model (
Figure 1) was verified as a potential method for predicting whether a compound obtained phloem mobility or not [
6,
7,
8,
9,
10]. For instance, using the Kleier model, N-carboxymethyl-3-cyano-4-(2,3-dichlorophenyl)pyrrole exhibits good phloem mobility [
10]. Furthermore, some compounds are absorbed by endogenous carriers in plants, such as glyphosate and paraquat [
11,
12] (
Figure 2). L-type amino acid transporters (LAT1/LAT2) play significant roles in the uptake of glyphosate [
11]. Paraquat uptake is involved in polyamine transporter RMV1 and AtPDR11 [
12]. Therefore, another approach to converting nonmobile pesticides into phloem-mobile types consists of introducing endogenous plant substances, such as glucose and amino acid peptides, to modify pesticide molecules by click chemistry [
13,
14,
15,
16,
17,
18], which involves a carrier-mediated process. For example, coupling a non-phloem-mobile insecticide with glycine could improve phloem mobility with fipronil-glycine conjugates [
15] (
Figure 3). Amino acid carriers were found more efficient in translocating phenyl pyrrole conjugates than sugar carriers [
16]. Four amino acid transporters, RcLHT6, RcANT15, RcProT2, and RcCAT, may be involved in the glycine–fipronil coupling phloem transport [
17]. Thus, the phloem mobility of exogenous substances correlates with their own physicochemical properties and plants’ endogenous carriers.
Phenazine-l-carboxylic acid (PCA) is an antibiotic secreted by
Pseudomonas sp. M18. [
19,
20] PCA is a dual-function fungicide capable of the broad-spectrum inhibition of plant pathogens and promoting plant growth [
21,
22]. It has the characteristics of a broad-spectrum and a high-efficiency. Currently, PCA is registered as a new microbially sourced fungicide for rice in China and has been widely promoted. However, PCA does not have phloem mobility [
23,
24]. In our previous reports, we have developed a vectorization strategy coupling the PCA to amino acids based on Carrier-mediated theory, which successfully confers phloem mobility to PCA [
23,
24,
25,
26,
27,
28,
29]. The PCA was absorbed by the plants in the form of conjugates and then hydrolyzed by amide hydrolase to PCA [
29]. (
Figure 4). However, the phloem mobility of these couplings should be further improved [
23,
29]. Meanwhile, some interesting phenomena have been discovered. For example, based on the Kleier prediction model, the conjugates PCA-L-Tryptophan and PCA-L-Tyrosine (
Figure 5) should have an excellent diffusion through the membrane, and phloem mobility should be observed. Nevertheless, the experimental results of the phloem sap analysis violate the Kleier model. PCA-L-Tryptophan and PCA-L-Tyrosine were found to have no phloem mobility, but this may be due to the lack of relevant amino acid carriers [
24]. Amino acid carriers should more easily recognize
PCA-Gly to improve phloem mobility, but their phloem mobility was not as satisfactory as expected because they are more hydrophilic with a low diffusion through the membrane [
24]. Thus, the single Kleier model or Carrier-mediated theory cannot achieve a reliable explanation of the phloem mobility of all exogenous substances. In the present paper, a novel strategy of combining Carrier-mediated theory and the Kleier model is proposed for the first time to improve compounds’ phloem mobility. On the one hand, based on Carrier-mediated theory, the active ingredient-amino acid conjugate operates as the molecular model; on the other hand, the N-alkylated amino acid conjugate improves the physicochemical properties by following the Kleier model to promote phloem mobility. Then, the capacity of the Kleier model and Carrier-mediated theory to design phloem-mobile pesticides is inspected, which may provide a more accurate and highly efficient basis for guiding the design and development of phloem-mobile pesticides.
To verify this strategy, PCA-glycine conjugate [
24] (a compound with phloem mobility synthesized by our research group) was chosen as the molecule model due to the glycine-rich nature of the model plant. Furthermore, a series of the N-alkylated derivatives of
PCA-Gly were designed and synthesized (
Scheme 1). Hydrogen linked with a nitrogen atom is substituted by methyl, ethyl, isopropyl, tert-butyl, and phenyl (
4a–
4f). Among them, the Glycine fragments guarantee that they can be carried by carriers, and the N-alkylated derivatives will enhance the hydrophilicity via a higher diffusion through the membrane. The phloem mobility of all the coupling compounds was evaluated by ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS) using castor bean seeds (
R. communis L.) and a castor bean plant model. The relationship between the movement of phloem with the structure of exogenous compounds was discussed by the Kleier model and Carrier-mediated theory.
3. Materials and Methods
3.1. Chemicals
All reagents and solvents were purchased from commercial suppliers. The melting point was determined by a WRR-Y melting point apparatus (Shanghai Yidian Physical Optical Instrument Co., Ltd., Shanghai, China). Thin-layer chromatography (TLC) was conducted on silica gel plates (GF254) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and spots were visualized on a ZF-I ultraviolet analyzer (Shanghai Gucun Electro-optical Instrument Factory, Shanghai, China). Column chromatography purification was carried out on silica gel (200–300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Nuclear magnetic resonance (NMR) spectra were obtained using an AVANCE III HD 400 NMR spectrometer (Bruker Corporation, Basel, Switzerland). Mass spectrographic analysis was conducted on a Thermo Scientific Q Exactive TM (Thermo Fisher Scientific, Waltham, MA, USA).
3.2. Plant Materials
Castor bean seeds (Ricinus communis L.) were provided by the Zibo Agricultural Science Research Institute. The castor seedlings were planted as previously reported (Yu et al., 2018). Then, 6-d-old seedlings were selected for the next experiments.
The adult castor bean plants were obtained according to methods described in a previous study [
32]. Castor seedlings were grown in nutrient soil in a greenhouse (25–30 °C, natural light) for 3–4 weeks until 3–4 leaves appeared, and cotyledons and primary leaves were removed.
3.3. General Synthesis Procedure for Title Compounds 3a–3l and 4a–4f
The synthetic route is described in
Scheme 1.
3.3.1. General Procedure for Glycine Ester Derivatives 1
As shown in
Scheme 1, A mixture of R
1NH
2 (1 mmol), BrCH
2COOR
2 (2 mmol), and K
2CO
3 (3 mmol) in DMF (15 mL) was stirred at room temperature for 12 h. Subsequently, 100 mL of water was added to the reaction mixture, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum [
33,
34].
3.3.2. Synthesis of Phenazine-1-Carbonyl Chloride 2
Phenazine-1-carboxylic acid (2 mmol) was dissolved in 20 mL of anhydrous CH
2Cl
2; then, oxalyl chloride (3 mmol) was slowly added. The reaction was stirred at reflux temperature for 8 h. The reaction solution was evaporated under vacuum, and the residue was dissolved in 15 mL anhydrous CH
2Cl
2, which was immediately used for the following reaction [
28].
3.3.3. General Procedure for PCA-Glycine Ester Derivatives 3a–3l
The glycine ester derivative 1 (2 mmol) was dissolved in CH
2Cl
2 at 0 °C, triethylamine (10 mmol) was added, and the reaction was stirred for 15 min. Then, phenazine-1-carbonyl chloride 2 (2 mmol) completely dissolved in 15 mL of anhydrous CH
2Cl
2 was added dropwise with respect to the above reaction system. The mixture was stirred at 0 °C for about 6 h until the reaction was complete (monitored by TLC). The reaction solution was washed with a 5% sodium hydrogen carbonate solution and extracted with CH
2Cl
2. Then, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuum. Finally, pure target compounds
3a–
3l were obtained by column chromatography (PE/EtOAc,
v/
v = 4:1) [
25].
3.3.4. General Procedure for PCA-Glycine Derivatives 4a–4f
Lithium hydroxide (10 mmol) was added dropwise to a solution of compound
3a (2 mmol) in water (10 mL) and 1,4-dioxane (10 mL), and the reaction mixture was stirred at room temperature for 5 h until the reaction was complete (monitored by TLC). The 1,4-dioxane and water were removed under vacuum, and the remaining solid was dissolved with a small amount of water. The pH of the aqueous solution was adjusted to 2 with 1 mol/L of HCl. The solid precipitate was then filtered and dried to obtain the pure target compound
4a. Compounds
4b–
4f were also synthesized by this method [
24].
3.4. Sap Collection from R. communis L. Seedlings
The method of phloem sap collection was the same as that recently described [
2,
24]. The cotyledons were immersed in a buffered solution containing 200 μmol/L test compounds, and roots were immersed in 500 μmol/L CaCl
2 solution. After 2 h of incubation, the hypocotyls were cut for phloem exudation. Phloem sap was collected at a 1 h intervals for 5 h. A series of standard solutions (1, 2, 5, 10, and 20 μmol/L) of the test compounds were prepared in methanol for calibration curves. The linear equations of test compounds are shown in
Table 5.
3.5. Phloem Mobility in Adult Castor Bean Plants
The methodology used for this phase is as follows. Prepare 1 M, 2 M, and 5 M liquid containing the compounds, wrap the upper two true leaves, stem, and matrix soil surface of the castor plant with cling film to avoid contamination of the liquid, and slowly smear the liquid on the lower two true leaves of the castor plant several times with a brush. The amount of liquid medicine used was 1 g, and the castor plants were exposed to natural light in the greenhouse. Repeat the above steps 3 times. Castor root was collected at 3 h, 6 h, 12 h, 18 h, and 24 h and stored at −20 °C for testing.
The pretreatment method of castor samples is as follows. Wash, dry, and section the castor roots. Add 50 mL of methanol with masher crush, add 30 mL of methanol wash segment, transfer to the triangle in the bottle, and seal it in plastic wrap. Conduct an ultrasonic extraction for 30 min, vacuum suction filter, filter residue with an appropriate amount of methanol and ultrasonicate for 10 min, vacuum suction filter again, combine the filtrate, and place the concentration in a rotary dryer until it is near dry to facilitate the following purification.
The purification procedure is as follows. The concentrated extract was transferred to a 250 mL separating funnel with a small amount of dichloromethane; then, 50 mL 10% sodium chloride solution and 5 mL NaOH solution were added. After mixing, 50 mL, 40 mL, and 30 mL dichloromethane was added separately, the extraction was shaken three times, and the lower layer (dichloromethane) was discarded. The pH of the alkaline aqueous phase was adjusted to 3 with 1.6 mL of glacial acetic acid (purity ≥ 99.5%); then, the dichloromethane phase was extracted with 50 mL, 40 mL, and 30 mL dichloromethane three times by shaking, and the dichloromethane phase was collected. After being dehydrated by anhydrous sodium sulfate, the dichloromethane phase was dried by rotation, and the volume was fixed with 5 mL of chromatographic methanol and filtered through a 0.45 μm membrane. A series of standard solutions (0.5, 1, 2, 5, 10, and 20 μmol/L) of test compounds were prepared in methanol for calibration curves. The linear equations of test compounds are shown in
Table 6.
3.6. Analytical Methods
The phloem sap was diluted with pure water (phloem sap/pure water, v/v = 1:9), and analyzed by ultra-high performance liquid chromatography mass spectrometer (UHPLC-MS) (Thermo UltiMate 3000 TSQ-Quantis, Waltham, MA, USA). A C18 reversed-phase column (3 um, 100 × 2.1 mm, Thermo Fisher Scientific Co., Ltd., MA, USA) was used for separations at 30 °C. The mobile phase was composed of methanol and water containing 0.1% formic acid with an isocratic elution (methanol/water containing 0.1% formic acid, v/v = 70:30) at a flow rate of 0.4 mL/min. And the injection volume was 10 μL. The optimized parameters of electrospray ionization in the positive mode were as follows: pos ion spray voltage, 3500 V; sheath gas, 30 Arb; aux gas, 5 Arb; ion transfer tube temp, 350 °C; and vaporizer temp, 400 °C.
4. Conclusions
All of the hydrolyzed compounds (
4a–
4f) with exposed carboxyl groups exhibited excellent phloem mobility in
R. communis L. compared to the non-phloem-mobile PCA and PCA-amino acid ester conjugates. The phloem mobility of
4a–
4c was significantly enhanced—8 to 10 times higher than
PCA-Gly. Therefore, the N-alkylation of
PCA-Gly promotes phloem mobility. Our previous studies have demonstrated that the carboxyl group is an amino acid-carrier binding site [
23,
24,
25,
26,
27,
28,
29]. Based on Carrier-mediated theory, N-alkylated amino acid conjugates will increase molecular width and the steric hindrance, resulting in the decrease in the carrier-binding conjugates. Compounds
4a–
4c are still within the binding range; thus, their phloem mobility increases with an increasing lipophilicity and exhibit the synergism of Carrier-mediated theory and the Kleier Model. The synergistic effect began to weaken starting with compound
4d. The
R. communis L. results indicate that small substituents can significantly improve PCA’s phloem mobility, and this can be quantified to enable a LogKow between 1.2 and 2.5. Compound
4e is difficult to combine with amino acid carriers due to the considerable steric hindrance of phenyl. Even if the lipophilicity was improved, the movement of the phloem is lower than
PCA-Gly. Compound
4f and
PCA-Gly are more hydrophilic and exhibit a small degree of migration in plants. The experiment involving the phloem mobility in adult castor bean plants showed that most of the tested compounds can pass through the wax layer and move in the phloem. This synergism is similar to that of
Ricinus communis L. Therefore, we suggest introducing plant endogenous compounds as a promoiety to improve the phloem mobility of pesticides via Carrier-mediated theory. It is necessary to consider the improvement of the physicochemical properties according to the Kleier model. This study verifies that the carrier-mediated theory and Kleier model can play a synergistic role in promoting the phloem transport of exogenous compounds. As far as we know, this theory is the first to combine the Kleier model with the Carrier-mediated theory in the design of phloem-mobile pesticides. We provide an active ingredient-amino acid conjugate structural model, which can also extend to other plant endogenous nutrients, such as glucose, peptides, etc. However, more data are still needed for supplements, which will be further studied. This research and its further iterations will contribute to a scientific theory for developing phloem-mobile pesticides.