Synthesis Of Amino Acid Guided Pesticides Based On Modifications Of The Cyano Group Of Fipronil And Study On Their Phloem Mobility

The "amino acid guided pesticides" are pesticides utilizing certain active transporter to achieve optimal distribution in plants by conjugating endogenous amino acid with the structure of pesticides.Based on this,a new series of amino acid-fipronil conjugates based on derivatization of the cyano group at the 3-position of pyrazole ring of fipronil was designed and synthesized(4a-l).All structures were confirmed via 1H,13 C NMR spectroscopy and ESI-HRMS spectrometry.The phloem mobility of new amino acid-fipronil conjugates was measured by R.communis seedlings system under the same experimental conditions using high performance liquid chromatography(HPLC).Experiments using Ricinus communis seedling system showed that all tested conjugates were phloem mobile except isoleucine-fipronil conjugate.It suggested that the cyano group-coupled amino acid fragment at the 3-position of the pyrazole ring of fipronil is feasible to acquire phloem mobility of the non-phloem-mobile parent compound.In general,the phloem mobility of compounds that have a small substituent at the ?-position of the amino acid fragment was better than that of the ones containing branched alkyl groups,aromatic rings,or heterocycles.Among them,conjugate 4g(serinefipronil)showed the highest phloem mobility,with the concentration in phloem sap being 52.00 ± 5.80 ?M.The phloem loading mechanism of new amino acid-fipronil conjugates were studied via physicochemical properties prediction,RT-q PCR analysis and Xenopus oocyte uptake experiments.The possible causes of differences in the phloem mobility of different amino acid-fipronil conjugates were explored in plant physiological level,transcription level and cell level.According to the prediction with Kleier model,seven of the conjugates(4a,4b,4c,4f,4g,4k and 4l)were located in the phloem-mobile area,five of the conjugates(4d,4e,4h,4i and 4j)were located in the non-phloem-mobile area.Conjugates 4g,4a,4d and 4e(the maximal and minimal phloem mobility inside or outside the phloem-mobile area of Kleier map respectively)were chosen to be the candidate conjugates for further study.Real-time Quantitative PCR results showed only the expression of Rc ANT15 was upregulated compared to the DMSO control after treatments with four candidate conjugates.Therefore,Rc ANT15 has a significant correlation with the uptake of these candidate conjugates.Xenopus oocytes uptake experiments demonstrated that Rc ANT15 was involved in the uptake of conjugates 4a,4g and 4d,except 4e.All in all,the castor bean amino acid transporter Rc ANT15 has the function of transporting conjugates 4g,4a and 4d,and the phloem loading process of conjugates 4g and 4a involved both passive diffusion and amino acid carrier system(Rc ANT15).This study provides a feasible and efficiency strategy that conjugating amino acids to existing non-phloem-mobile pesticide structure to acquire phloem mobility,and provides practical experience and theoretical guidance for development of novel amino acid guided pesticides.