Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC Nowadays, it is being commercialized worldwide as Movento, Ultor, or Kontos for insect pest control in a large variety of crops, such as stone fruits, pome fruits, berry fruits—including grapes—, citrus fruits, nuts, vegetables, etc. Originally derived from the natural antibiotic thiolactomycin, SP is structurally characterized by a cyclohexane ring spiro-linked tetramate core with three substituents: a methoxy, an ethoxycarbonyl, and a dimethylphenyl ring  so it can move along the phloem and the xylem, thus allowing to combat pests that are difficult to reach, such as the grape mealybug (Planococcus ficus)6. Further metabolism may occur in some crops, such as reduction or hydroxylation of the enol-carbonate double bond of SP-enol to form spirotetramat-monohydroxy (SP-mono) or spirotetramat-ketohydroxy (SP-keto), respectively. A fourth metabolite can occasionally be generated by glycosylation of the hydroxyl moiety, thus forming spirotetramat-enol-glucoside (SP-glu)7. SP does not show severe toxicity in mammals, though it may cause skin-sensitization and can be an eye irritant8. For those reasons, different residue definitions have been adopted by official regulatory agencies according to the target commodities and to the analytical purposes. In 2008, the Joint FAO/WHO Meeting for Pesticide Residues established the residue definition for monitoring the compliance with the maximum residue limits (MRL) for plant commodities as the sum of SP and SP-enol, expressed as SP9, and this definition was also adopted by the European Commission10.Spirotetramat (SP), also known as BYI08330, was developed by Bayer CropScience a few years ago, and it was approved as a pesticide in the European Union and in the USA in 201413. Additionally, the QuEChERS method  has been set out as the most recommendable approach for residue extraction and sample preparation15. Zhu et al. optimized the analysis of SP residues by QuEChERS extraction and HPLC‒MS/MS in fruits and vegetables with limits of quantification (LOQ) in the low µg/kg range16. In 2015, Mohapatra et al. published the evaluation of SP and SP-enol in grapes by GC‒MS with a LOQ of 50 µg/kg17. Moreover, Berset et al. reported the determination of systemic insecticides, including SP, in commercial red and white wines by HPLC‒MS/MS with a LOQ of 1 µg/L18.Current analytical methods for SP residue determination in food commodities include high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled to mass spectrometry (MS) or other detection methods19. In the present study, monoclonal antibodies (mAbs) to SP and SP-enol were raised for the first time with the aim to develop alternative immunoassays for simultaneous residue appraisal in food samples. In order to generate high-affinity mAbs with adequate specificity, a new immunizing hapten displaying the characteristic cyclohexane ring spiro-linked tetramate core was prepared. Moreover, an additional hapten was designed in order to obtain heterologous assay conjugates that could improve assay sensitivity and conjugate stability. Our goal was to optimize and validate different bioanalytical methods—a competitive enzyme-linked immunosorbent assay (cELISA) and a lateral flow immunochromatography assay—for total SP and SP-enol determination in relevant foodstuffs, such as grapes and their most commonly processed commodities, i.e., juice and wine.Nowadays, bioanalytical methods are being adopted as complementary screening tools for the rapid determination of chemical contaminants. Efficient sample preparation procedures and more robust and precise results have paved the way for the implementation of antibody-based methods, both in official analytical laboratories and in quality control departments of other stakeholders of the food chain. The high sensitivity, portability, and friendly use of immunochemical methods at competitive prices are being increasingly appreciated by the industrial sector. Recently, polyclonal antibodies to SP and SP-enol were generated showing the great potential of the studied synthetic strategy to prepare haptens20. The nature and position of the chemical groups of the hapten are important for binding, but also the affinity and specificity of the newly-formed antibodies are greatly determined by the conformation of the molecule, and consequently the position of the spacer arm is of great relevance21. Nevertheless, the optimum linker tethering site is difficult to predict, particularly when antibodies with broad specificity are searched. Taking into account these premises, haptens were designed to generate high-affinity antibodies to SP and its metabolite SP-enol, and to develop highly sensitive immunoassays  with the spacer arm at the aromatic ring of SP, which is opposite and complementary to that of hapten SPo, was designed. Additionally, with a view to developing immunoanalytical methods, and because hapten heterology has often been demonstrated as a good strategy to improve the performance of immunoassays, hapten SPh—a more stable variant of hapten SPc in which the labile carbonate group of the spacer arm was replaced by an ether bond—was conceived.Nobel prize-winner Karl Landsteiner demonstrated long ago the stereospecific binding behaviour of antibodies to haptensm was prepared by total synthesis from aryl iodide 1, which in turn was obtained, as detailed in the Supplementary Information file  acetic acid. This procedure followed a synthetic route similar to that previously used for the preparation of the methylated analogue at the C-5 position of the phenyl ring22. First, O-methylation of the hydroxyl group at the C-8 position of the 1-azabicyclic moiety was carried out . The incorporation of the carboxylated spacer arm as its tert-butyl ester was based on the Sonogashira cross-coupling reaction between the iodinate position of 2 and the terminal alkyne-ester 3, which was previously prepared from commercially available hex-5-ynoic acid23. The cross-coupling reaction was carried out efficiently using Cl2Pd(PPh3)2 as the source of the palladium catalyst, CuI as co-catalyst, and Et3N as the base, in DMF at room temperature. The incorporation of the carboxylated aliphatic linear chain was completed from the alkyne resulting from the cross-coupling reaction, i.e. 4, by hydrogenation reaction of the triple bond under homogeneous conditions using Wilkinson's catalyst. The synthesis of hapten SPm was readily completed from intermediate 5 by cleavage of both the methoxy group—to regenerate the unsubstituted amide group—and the tert-butyl ester group—to form the corresponding carboxylic moiety. Thus, demethylation of the methoxy-2H-pyrrole ring of 5, followed by acid catalysed cleavage of the tert-butyl ester group, afforded the target hapten SPm in an excellent global yield. Overall, the synthesis of hapten SPm from aryl iodide 1 proceeded in 5 steps with a total yield of ca. 40%.Hapten SPe Figure , from 1,h, which incorporates the spacer arm through a C‒O ether linkage at the enolic oxygen atom, was significantly simpler than that of hapten SPm . A much higher yield was found under non-basic conditions using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) in dry acetonitrile at room temperature for 24 h. Under these conditions, the N-hydroxysuccinimidyl ester SPm-NHS was obtained in 80% yield  of the newly prepared bioconjugates are provided in the Supplementary Information file.BSA, ovalbumin (OVA), and horseradish peroxidase (HRP) were employed as carrier proteins to prepare bioconjugates with the corresponding active esters of haptens SPm—named SPm#23, SPm#25, and SPm#216—and three from conjugate BSA‒SPo—named SPo#227, SPo#237, and SPo#243. These antibodies contained κ light chains and were of the IgG1 isotype, except mAb SPm#25 which was IgG2a. The affinity to SP and SP-enol was studied by checkerboard cELISA in the capture antibody-coated direct format, using homologous and heterologous enzyme tracers . SP and SP-enol standards were prepared in PBS  and tracer solutions for the competitive step were prepared in PBS-T (PBS containing 0.05% (v/v) Tween-20). Under these conditions, Amax values higher than 0.8 were achieved with four antibodies  combined with the homologous enzyme conjugate . Interestingly, a very low IC50 value (0.55 nM) for SP-enol was obtained with mAb SPm#23 combined with HRP‒SPh heterologous tracer.Six mouse mAbs were obtained; three were from immunogen BSA‒SPm and BSA‒SPo) afforded equivalent results. Binding to both SP and SP-enol was observed with the two types of antibodies, and SP-enol was always better recognized. The partial in vivo hydrolysis of the carbonate group of haptens SPm and SPo during immunization could explain this result. Cross-reactivity with the main SP metabolites was studied with antibody SPm#23. The affinity of this mAb to SP-enol was at least 10 and 20 times higher than to SP and SP-glu, respectively—binding to SP-keto and SP-mono was almost negligible  Tween-20. It was observed that the studied immunoassay was quite tolerant to pH changes between pH 6.0 and 8.5  and the latter will be used to extract solid samples (grapes). Standards were prepared in the studied solvent dilutions in MilliQ water, and tracer solutions for the competitive step were prepared in 20 mM phosphate buffer, pH 7.4, containing 280 mM NaCl and 0.05% (v/v) Tween-20. It was observed that none of the evaluated solvents was well tolerated by the selected immunoassay . For the competitive step, enzyme tracer solutions were prepared in TBS-T  Tween-20). As shown in the inset of Table 4. Controls without the hydrolysis step were also evaluated. Finally, the contents of SP, SP-enol, SP-keto, and SP-mono in the organic extracts were quantified —under the optimized conditions. The Amax value of the standard curve was near 1.0, the slope was − 1.4, and the background signal was negligible. The calculated limit of detection (LOD) value was 0.02 μg/L, and the dynamic working range was between 0.05 and 0.31 μg/L. Moreover, excellent intra-day and inter-day precision values (below 10%) for the Amax and IC50 values were observed.The sigmoidal inhibition curve obtained for SP-enol and the final assay conditions of the developed direct cELISA are provided in Table max and IC50 values are depicted in Figure max value was mostly acceptable even at a low dilution of the samples, except for the red wine, and the deviation of the IC50 value was acceptable or just slightly below − 20% at a fivefold dilution factor for all of the assayed food samples.The performance of the developed immunoassay to analyse SP and SP-enol in wine, grape juice, and grape extracts was assessed. Solid samples were extracted by the QuEChERS method. To study the matrix effects, SP-enol standard solutions were prepared in wine, juice samples, and organic extracts, and each solution was diluted to a different extent with 40 mM NaOH. After 10 min, samples were 1:1 diluted with dilution buffer, and wine and juice samples were cleaned-up with polyvinylpolypyrrolidone (PVPP). The enzyme tracer solution was prepared in TBS-T. The matrix effects of wine, grape juice, and grape extracts over the ASP-free samples were fortified between 2.5 and 500 μg/L with SP, SP-enol, and a 1:1 mixture of both compounds. Samples were diluted with 40 mM NaOH solution and incubated, and then 1 volume of dilution buffer was added. Wine and juice samples were cleaned-up with PVPP. Recovery values were determined from a SP-enol standard curve run in the same microtiter plate. Excellent recovery values between 80 and 120%, and coefficients of variation below 20% were obtained with the developed direct cELISA  for the control line, and gold nanoparticle (GNP)-labelled antibody SPm#23 in the mobile phase. To optimise the amount of GNP conjugate as well as the pH and ionic strength of the assay buffer, tests were run with different standard concentrations and a blank. SP-enol standard solutions were 0.1 and 0.3 μg/L and SP standard solutions were 0.3 and 1.0 μg/L. The immunoassay response was determined as the quotient between the signal of the test line (T) and the signal of the control line (C), and the inhibition rate was calculated. Figure S8 shows the T/C ratios and inhibition rates obtained with varying amounts of GNP‒mAb conjugate. The highest inhibition rate was obtained with the lowest assayed quantity of bioconjugate (5 µL); however, the T and C values were too low for reading with the naked eye. Therefore, 10 µL of GNP‒mAb conjugate was chosen as the optimum volume for a total of 100 µL of reaction mixture. Additionally, the pH and the ionic strength were optimized for this lateral flow immunoassay . The inhibition rate was not significantly changed at pH 7.5, 8.0, and 8.5, whereas a somewhat higher inhibition rate was found at 60 mM NaCl concentration. Consequently, 100 mM Tris·HCl buffer, pH 8.0, containing 60 mM NaCl and 0.05% (v/v) Tween-20 was chosen as assay buffer for immunochromatographic analysis.A lateral flow immunochromatography assay was optimized and characterized using BSA‒SP27. The 2014 EU Regulation 519/2014/EU for validation of semi-quantitative screening analytical methods with inversely proportional response was followed28. Two white wine and two red wine samples were fortified with SP or SP-enol and fivefold diluted in 40 mM NaOH solution. After 10 min incubation at room temperature, they were 100-fold diluted with assay buffer. Each sample was measured twice every day during 5 consecutive days (n = 20). The European MRL for SP in wine grapes is 2000 μg/kg, but no MRL has been set so far for wines. Nevertheless, an average transfer factor from grapes to wine of 0.5 has been previously reported by EFSA (European Food Safety Authority)10. Consequently, the screening target concentration (STC) for SP in this study was set at 1000 μg/L. Figure t-value of 1.7291 for a 5% ratio of false negative results at the STC, the obtained cut-off values were 0.5 for the two types of wines. Moreover, the rates of false suspect results—blank samples that could be classified as suspect—were below 2% for both wines  as solvent, and kept at − 20 °C. Technical SP and formulated SP (Movento Gold 10% SC) were kindly provided by Bayer CropScience.Chemicals and apparatus, as well as general experimental techniques that were employed for the synthesis of novel haptens, for the preparation of bioconjugates, for hybridoma generation, and for immunoassay development are listed and described in the Supplementary Information file. SP -1-azaspiro[4.5]dec-3-en-4-yl carbonate; CAS registry number 203313–25-1; MW 373.45), SP-enol, SP-keto, SP-mono, and SP-glu analytical standards, Pestanal grade, were purchased from Merck. Pesticide stock solutions were prepared in amber glass vials using o was reported in a previous study19. Details of all of the synthetic steps and characterization data of intermediate compounds are provided in the Supplementary Information file.All reactions involving air-sensitive compounds were conducted in oven-dried glassware under a nitrogen atmosphere. The synthesis of hapten SPm was obtained in five synthetic steps as a semisolid  3400‒2600 (m), 3220 (m), 2938 (s), 2859 (m), 1779 (s), 1714 (s), 1214 (s), 1100 (m), 756 (m), 734 (m); 1H NMR  δ 7.83 , 7.12 , 7.03 , 6.95 , 3.99 , 3.38 , 3.23 , 2.55 , 2.31 , 2.26‒2.13 , 2.22 , 1.92 , 1.81‒1.71 , 1.71‒1.53 , 1.50‒1.42 , 1.42‒1.31 , 1.09 ; 13C NMR  δ 178.5 (C-1), 171.3 (NCO), 165.4 (C-4′), 150.0 (OCO2), 139.8 (C-1 Ph), 134.5 (C-3 Ph), 130.4 (C-5 Ph), 129.5 (C-2 Ph), 129.0 (C-6 Ph), 127.8 (C-4 Ph), 121.6 (C-3′), 77.5 (C-8′), 65.9 (CH3CH2OCO2), 60.9 (C-5′), 55.9 (OCH3), 35.2 (C-6), 34.3 (C-2), 31.7 (C-6′/C-10′), 31.1 (C-5), 28.7 (C-4), 28.5 (C-7′/C-9′), 24.8 (C-3), 19.4 (CH3-Ph), 13.9. (CH3CH2OCO2); HRMS (TOF–MS ES+) calcd for C26H36NO7 [M + H]˙+ 474.2486, found 474.2484.Hapten SPlid Fig. b. IR νmah was obtained from SP-enol in two synthetic steps  3265 , 2942 (m), 2868 (w), 1702 (s), 1685 (s), 1648 (s), 1337 (s), 1104 (s), 935 (w), 813 (w); 1H NMR  δ 7.69 , 7.07 , 7.01 , 7.00 , 3.73 and 3.62 , 3.38 , 3.24 , 2.29 and 2.16 , 2.26 , 2.22‒2.12 , 1.99‒1.84 , 1.70‒1.60 , 1.58‒1.35 , 1.31‒1.19 ; 13C NMR  δ 178.0 (C-1), 174.4 (NCO), 173.3 (C-4′), 135.0 (C-5 Ph), 134.9 (C-1 Ph), 132.0 (C-6), 130.5 (C-2), 129.8 (C-3 Ph), 129.3 (C-4 Ph), 106.0 (C-3′), 77.8 (C-8′), 71.4 (C-6), 60.9 (C-5′), 56.0 (OCH3), 34.1 (C-2), 32.8 and 32.2 (C-6′ and C-10′), 29.1 (C-5), 28.5 and 28.6 (C-7′ and C-9′), 25.2 (C-4), 24.4 (C-3), 21.0 and 19.7 (2 × CH3-Ph); HRMS (TOF–MS ES+) calcd for C24H34NO5 [M + H]+ 416.2431, found 416.2442.Hapten SPeps Fig. c. IR νmam and SPh were activated through formation of the corresponding N-hydroxysuccinimidyl ester using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) in dry acetonitrile  from 8.0 mg of hapten SPm (80% yield). 1H NMR  δ 7.13 , 7.04 , 6.96 , 6.29 , 3.99 , 3.39 , 3.24 , 2.83 , 2.59 , 2.57 , 2.23 , 2.26‒2.16 , 1.93 , 1.82‒1.71 , 1.67‒1.53 , 1.50‒1.29 , 1.08 .SPh-NHS ester: 10.9 mg (26.2 µmol) from 12 mg of hapten SPh (89% yield). 1H NMR  δ 7.08 , 7.02 , 7.00 , 5.93 , 3.72 and 3.62 , 3.39 , 3.25 , 2.83 , 2.56 , 2.29 and 2.17 , 2.25‒2.18 , 1.93 , 1.71–1.58 , 1.56–1.46 , 1.41–1.26 .o was described in a previous article19. Concerning SPm and SPh conjugates, compounds SPm-NHS and SPh-NHS were dissolved in DMF (50 mM) and coupled to BSA, OVA, and HRP in 100 mM phosphate buffer, pH 7.4, as described in the Supplementary Information file. A small aliquot of each conjugate solution was dialysed using Slide-A-Lyzer MINI dialysis units from Thermo Scientific. Hapten density of protein conjugates was determined by MALDI-TOF‒MS analysis in positive linear mode (1500 shots for each position) in a mass range of 10,000‒120,000 m/z, as described in the Supplementary Information file.Preparation of conjugates of hapten SPm and the other with conjugate BSA‒SPo. Experimental design of the immunization procedures was approved by the Bioethics Committee of the University of Valencia. The European Directive 2010/63/EU and the Spanish laws and guidelines (RD1201/2005 and 32/2007) were followed for animal manipulation. Monoclonals were purified from late stationary-phase culture supernatants by salting out with ammonium sulphate and subsequent affinity chromatography, and they were stored as ammonium sulphate precipitates at 4 °C. Further details can be found in the Supplementary Information file.Two groups of four 8-week old female Balb/c mice were intraperitoneally immunized; one with conjugate BSA‒SPMicroplates were precoated with GAM in order to immobilise the mAb. The competitive step was carried out by mixing the analyte solution or diluted sample with the enzyme tracer solution. Standard curves were built in borosilicate glass tubes from 0.03 to 30 nM by a threefold serial dilution of the most concentrated standard solution, and a blank  was also run. Concentrated stock solutions of the analytes in anhydrous DMF were used to prepare the most concentrated standard solution—the DMF concentration was always lower than 0.1% (v/v). The complete immunoassay procedure can be found in the Supplementary Information file.SP, SP-enol, SP-keto, and SP-mono standard solutions were prepared by serial dilution in acetonitrile from 1 to 2500 μg/L in amber glass vials and kept at − 20 °C. The injection volume was 5 µL and the column temperature was 45 °C. The analysis was carried out using a C18 reverse phase column with a gradient of 0.2% (v/v) formic acid in water (A) and acetonitrile (B) as mobile phase at a flow rate of 0.3 mL/min. The elution profile was 0‒3 min, from 5% B to 95% B, 3.0‒3.5 min, 95% B, 3.5‒3.6 min, from 95% B to 5% B, and 3.6‒8 min, 5% B. Spectra were acquired in positive ionization multiple reaction monitoring mode with interchannel delay of 0.02 s.h conjugate and GAM, respectively. The GAM and BSA conjugate solutions—at 1 and 0.5 mg/mL, respectively—were dispensed at 0.5 µL/cm. Then, the membranes were fixed on a 30 cm backing card, and the absorbent and sample pads were fixed. Finally, 4-mm strips were cut with the aid of a guillotine, and they were stored in opaque plastic tubes with desiccating agent at 4 °C. Colloidal GAM-coated GNP were diluted with 10 mM HEPES, pH 7.4, to OD 1.0, and 5 μL of monoclonal SPm#23 solution in BioStab was added over 1 mL of GNP dilution up to a final mAb concentration of 0.5 µg/mL. The mixture was incubated at room temperature during 1 h, and Tween-20 was added to reach a concentration of 0.05% (v/v). The obtained gold bioconjugate was stored at 4 °C.The test and control lines on the nitrocellulose membrane were prepared by applying BSA‒SPConcentrated stock solutions of the analytes in anhydrous DMF were used to prepare the standard solution at the highest concentration. SP and SP-enol standard curves were built from 0.01 to 10 µg/L by threefold serial dilution in buffer from the most concentrated standard, and a blank  was also run. The GNP‒mAb conjugate (10 µL) was mixed with the diluted sample or standard solution in buffer (90 µL) in a microplate well, and the immunochromatographic strip was immediately inserted into the well. The chromatography was allowed to proceed vertically, and after 10 min at room temperature the sample pad was detached from the strip to stop the flow. Then, the strips were dried with a gentle cold air current.17. Briefly, 15 mL of acetonitrile was added to 15 g of homogenized sample in a 50-mL polypropylene tube. The mixture was vigorously stirred during 1 min with the aid of a vortex stirrer, and 1.5 g of sodium acetate and 6.0 g of anhydrous magnesium sulphate were added. The mixture was stirred again for 2 min. After centrifugation for 10 min at 2200×g, 12 mL of the organic phase was transferred to a 50-mL tube containing 600 mg of primary-secondary amines and 1.8 g of anhydrous magnesium sulphate. The mixture was vortexed during 1 min and centrifuged 10 min at 2200×g. Finally, the organic extracts were kept at − 20 °C. Grape extracts were treated with 40 mM NaOH solution during 10 min at room temperature before cELISA analysis. Then, samples were 1:1 diluted in dilution buffer .Grapes from local markets were homogenized with a mixer and extracted using the QuEChERS methodology as described by Mohapatra et alWine and grape juice samples from local supermarkets were diluted in a 40 mM NaOH solution and incubated 10 min at room temperature. The mixtures were 1:1 diluted in dilution buffer, and a 1-mL aliquot was cleaned-up with 30 mg of PVPP by strong stirring with the aid of a vortex during 2 min. Then, samples were centrifuged at maximum speed during 5 min with a microtube centrifuge, and the supernatant was collected for immunoanalysis.50) of the maximum absorbance (Amax) obtained in the absence of analyte. The LOD of the assay was defined as the IC10 of the sigmoidal inhibition curve. The LOQ was experimentally determined as the lowest analyte concentration that provided accurate and precise results using fortified food samples.The absorbance of each microplate well was immediately read at 492 nm using a reference wavelength at 650 nm, and values were processed with KCJunior software from BioTek Instruments. The SigmaPlot Version 14.0 was used to fit the experimental values to a standard four-parameter logistic curve. Immunoassay sensitivity was estimated as the concentration of analyte affording a 50% decrease  signal of control and test lines of immunostrips were read with a digital scanner, and data were processed with the ImageJ software from the US National Institutes of Health. The T/C signal value was calculated from the quotient between the test line signal and the control line signal. The cut-off response value was determined according to the following formula for methods with inversely proportional response with the analyte concentration:Supplementary Information.


In Fig. 3B, the labels for the 3rd and 4th data sets previously labeled ΔspeE2+pSpeE2 and Δcluster+vector, respectively, were swapped. The correctly labeled figure appears below. There are no corresponding changes to the text or figure legend.Volume 11, no. 3, e00575-20, 2020,


The selected derivatives were synthesized and evaluated for their antioxidant and antimicrobial potential against several Gram (+ ve), Gram (−ve), as well as fungal strains. The results indicated that synthesized compounds exhibited good antioxidant, antimicrobial, and better preservative efficacy in food preparation as compared to the standard compounds. No significant differences were observed in different parameters as confirmed by Kruskal–Wallis test (p < 0.05). Docking results have been found in good correlation with experimental wet-lab data. Moreover, the mechanistic insight into the docking poses has also been explored by binding interactions of amygdalin derivative inside the dynamic site of G-6-P synthase.G-6-P synthase enzyme has been involved in the synthesis of the microbial cell wall, and its inhibition may lead to the antimicrobial effect. In the present study, we designed a library of amygdalin derivatives, and two most active derivatives selected on the basis of various parameters viz However, the existing preservatives have been associated with severe side effects such as estrogenic effect, breast cancer, contact eczema, endocrine disruptors and many other type cancers, etc. Hence, the researchers have been compelled to search for new, better, and safe preservatives5.Nowadays, several antimicrobial and antioxidant based preservatives such as p-hydroxybenzoates, parabens, benzalkonium chloride, dibromodicyanobutane, dimethyl dithiocarbamate, dimethoxy dimethyl hydantoin, formaldehyde, etc. are available in the market6. Hence, for pathogenic bacteria the inhibition of microbial cell wall synthesis may be used as a vital target to produce the antimicrobial effect. The probable target site for the inhibition of microbial cell wall synthesis includes PBPs (transpeptidases), β-lactamase, terminal D-Ala-D-Ala in Lipid II, and Glucosamine-6-Phosphate synthase (G-6-P synthase), etc.8.To attain this, the researchers have focused on discovery of novel mechanisms and target sites in addition to the reported mechanisms responsible for antimicrobial action. The different target site for the action of antimicrobials includes inhibition of the cell membrane synthesis, metabolic pathways (folic acid synthesis inhibition), protein synthesis, leakage from cell membrane, and interference with DNA and RNA replication, etc. The microbial cell wall provides mechanical support to microbes and regulates the diffusion processG-6-P synthase is a complex enzyme involved in the formation of Uridine diphosphate acetylglucosamine (UDP-GlcNAc) and catalyzes the first step in hexosamine biosynthesis. It converts Fru-6-P into Glucosamine-6-Phosphate (GlcN-6-P) using glutamine as the source of ammonia. GlcN-6-P is a precursor of Uridine diphosphate N-acetylglucosamine (UDP-NAG) from which other amino sugar-containing molecules are derived. One of these products, N-acetyl glucosamine, is an essential constituent of the peptidoglycan layer of bacterial and fungal cell wall.9. The availability of a three-dimensional crystal structure of G-6-P synthase (protein data bank id 1moq) shall be used to explore, and evaluate a large number of molecules to find out better inhibitors of G-6-P synthase.The molecular docking can screen thousands of molecules for their affinity towards a particular target site using various softwares10. Amygdalin, a cyanogenic glycoside and is present in variable amounts in seeds of fruits like apricot, peach, plum, etc. and fruits like nectarine, chokeberry, christmas berry, barley, brown rice, buckwheat groats, cherry, etc.12.A large number of plant-based extracts and phytoconstituents are available, which possess excellent antimicrobial activity; however, there is a lack of data for their preservative effectiveness if compared to the commercially available preservatives16.The pharmacological potential of amygdalin includes antitussive, antiasthmatic, digestive, anti-atherogenic, inhibition of renal interstitial fibrosis, prevention of pulmonary fibrosis, lung injury due to hypoxia, immune system regulation, antitumor, anti-inflammatory, keratoconjunctivitis sicca, emphysema, leprosy, vitiligo, antimicrobial and antiulcer etc.20. Hence, based on the available data, it was planned to explore the amygdalin derivatives for their G-6-P synthase inhibitory potential along with their antioxidant, antimicrobial, and preservative efficacy potential in food preparation.Some literature data also concluded that several natural moieties like gallic acid derivatives, ferulic acid, p-coumaric acid, ε-Polylysine, etc. have been evaluated for their preservative effectivenessS. aureus MTCC 3,160, P. aeruginosa MTCC 1934, E. coli MTCC 45, C. albicans MTCC 183, and A. niger MTCC 282 were purchased from MTCC, Chandigarh. Chemical reactions were monitored by TLC on silica gel plates in iodine and UV chamber. The Sonar melting point apparatus in open capillary tube was used for the recording of melting points. 1H NMR and 13C NMR spectra were confirmed in DMSO and deuterated CDCl3 on Bruker Avance II 400 NMR spectrometer at a frequency of 400 MHz downfield to tetramethylsilane standard. The FTIR spectra were recorded on Perkin Elmer FTIR spectrophotometer with the help of the KBr pellets technique. Waters Micromass Q-ToF Micro instrument was used for Mass spectrum recording, and elemental analysis was done by Perkin Elmer 2,400 elemental analyzer.All the chemicals required for experimental work were of analytical grade and were purchased from LobaChemie, SRL, and Sigma Aldrich. Nutrient agar, nutrient broth, sabouraud dextrose agar, and sabouraud dextrose broth required for antimicrobial and preservative efficacy were obtained from Hi-media Laboratories. Streptomycin, ciprofloxacin, ampicillin and fluconazole were obtained as a gift sample from Belco Pharma, Bahadurgarh. Microbial strains 21. The remained docking evaluations were subjected to a Monte Carlo procedure to minimize the energy score.The Schrodinger, Inc.  software Maestro 11 was used for the computational calculations and docking studies. Laboratory for Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, Rohtak, INDIA, was used for the computational work. The receptor-grid files were generated by grid-receptor generation program Glide, version 6.6, 2015. Grid-based ligand docking was used, which utilized the hierarchical sequence of filters to produce possible conformations of the ligand in the active-site region of the protein receptor. At this stage, raw score values and geometric filters were prepared out unlikely binding modes. The next filter phase involves a grid-based force field evaluation and refinement of docking experiments, including torsional and rigid-body movements of the ligandThe energy differences were calculated using the equation:22. Pdb id 1moq, having resolution 1.57 Å was selected based on the lowest resolution and availability. All the water molecules except metals coordinate and present between the ligand and protein were removed. The energy-restrained structure of the protein G-6-P synthase was constructed with the help of the Optimized Potentials for Liquid Simulations -2005 (OPLS-2005) force field.The X-ray protein structure coordinates of G-6-P synthase were downloaded from Protein Data Bank and were prepared with the help of the Schrödinger protein preparation wizard ‘Prepwiz’The three-dimensional structural library was prepared using the Chemdraw software and preceded for energy minimization using the LigPrep tool for the correction of coordinates, ionization, stereochemistry, and tautomeric structure to gain the appropriate conformation through the addition or removal of hydrogen bonds. The partial charges were computed according to the OPLS-2005 force field  at biological pH and used for molecular docking studies.The amygdalin derivatives were synthesized by using the procedure outlined in Scheme 1 by enzyme catalysis. Here, 40 ml of acetone containing 0.1 mol/L amygdalin and 0.3 mol/L of sinapic and syringic acid in 1 g of Novozyme 435 were taken in incubator shaker at 200 rpm at 45 °C (48 h). The filtration of the mixture terminated the reaction. Both the compounds in the series were synthesized according to the standard procedures as outlined in Scheme 1 : δ 8.69 , 7.50–7.43 , 7.41–7.32 , 7.32–7.24 , 6.82 , 6.28 , 5.80 , 5.00 , 4.76 , 4.71 , 4.59 , 4.40–4.30 , 4.33–4.25 , 4.12 , 3.90 , 3.81 , 3.70–3.56 , 3.56–3.47 , 3.45–3.24 , 3.16 ; 13C NMR  δ 168.20, 149.08, 146.99, 138.98, 134.91, 130.91, 130.11, 128.91, 126.93, 119.31, 115.50, 106.56, 104.04, 100.85, 76.13, 74.19, 74.10, 74.00, 73.12, 72.60, 71.10, 70.23, 68.91, 68.25, 63.67, 56.59; IR (KBr pellets): 1,029 cm−1 (–C–O–C), 1,074 cm−1 (–C–C–), 1,449 cm−1 (–C=C–), 1642 cm−1 (–C=N–), 2,875 cm−1 (–C–H–), 3,371 (–OH–); MS ES + (ToF): m/z 663.22 [M+ + 2]; CHNS: Calc (C31H37NO15): C, 56.11; H, 5.62; N, 2.11; O, 36.16; Found C, 56.14; H, 5.64; N, 2.10; O, 36.18.mp: 230–232 °C; TLC : Rf = 0.67; Yield = 69.55%; M.Wt. = 622.55; 1H NMR : δ 8.69 , 7.50–7.43 , 7.41–7.32 , 7.32–7.24 , 7.18 , 5.80 , 5.00 , 4.76 , 4.71 , 4.59 , 4.37 , 4.33–4.25 , 4.24 , 4.01 , 3.90 , 3.81 , 3.65 , 3.57–3.27 , 3.16 ); 13C NMR : δ 166.71, 147.85, 141.19, 134.88, 130.91, 130.11, 128.91, 120.59, 119.27, 106.79, 102.61, 101.58, 76.55, 75.99, 75.68, 75.49, 74.47, 73.99, 71.60, 70.81, 69.52, 68.85, 64.07, 56.35; IR (KBr pellets): 1,029 cm−1 (–C–O–C), 1,074 cm−1 (–C–C–), 1,449 cm−1 (–C=C–), 1642 cm−1 (–C=N–), 3,029 cm−1 (–C–H–), 3,371 cm−1 (–OH–); MS ES + (ToF): m/z 622.18 [M+ + 2]; CHNS: Calc (C28H32NO15): C, 54.02; H, 5.18; N, 2.25; O, 38.55; Found C, 54.05; H, 5.15; N, 2.27; O, 38.56.mp: 239–241 °C; TLC : RAntioxidant activity of the synthesized was evaluated photocolorimetric assay by using DPPH free radical scavenging method. Briefly, 0.1 mM solution of DPPH in methyl alcohol was prepared, and 1 mL of this solution was added to 3 mL of sample or standard. Discolorations were measured at 517 nm after incubation for 30 min at 30 °C in the dark. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The test was performed in triplicate and the % inhibition values of given samples was calculated by using the formula:c was the absorbance of the control, and As was the absorbance of the sample23.Here, AS. aureus MTCC 3,160, P. aeruginosa MTCC 1934, E. coli MTCC 45, P. mirabilis MTCC 3,310, C. albicans MTCC 183, and A. niger MTCC 282 by using the tube dilution method. Dilutions of test and standard compounds were prepared in double strength nutrient broth I.P. (bacteria) or sabouraud dextrose broth I.P. (fungi)24. The slants of E. coli, P. aeruginosa, P. mirabilis, and S. aureus were incubated at the 30–35 °C for 24 h. The slants of C. albicans were incubated at 20–25 °C for 48 h, whereas; the slants of A. niger were incubated at 20–25 °C for 5 days. After the incubation period sterilized 0.9% NaCl solution was used to harvest the bacterial, and fungal cultures from agar slant through proper shaking and then the suspensions of microorganisms were diluted with the sterile 0.9% NaCl solution to Colony Forming Unit (CFU) count was adjusted by adjusting the density of microorganism suspension to that of 0.5 McFarland standards by adding distilled water. The number of CFU was determined by dilution pour-plate method. A serial dilution of 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.62 µg/mL was used for determination of MIC. The samples tubes were incubated at 37 °C for 24 h (bacteria), at 25 °C for 7 days (A. niger), and at 37 °C for 48 h , and the results were recorded in pMIC25.The antimicrobial activity of the synthesized compounds was determined against 26.The selected most active antioxidant/antimicrobial compounds were further evaluated for their preservative potential in the cosmetic product, White lotion USP and food products; such as fresh aloe vera juice the cosmetic product as per the procedure mentioned in USP 200427.Aloe vera leaves were cleaned with distilled water and cleaned with 0.5% chlorine water. The leaf base and tip were chopped 1.5 inches and 3 inches, respectively. Margins of leaves were removed with the help of a stainless steel knife. The pulp was washed 2–3 times with distilled water to remove the exudates and homogenized with the help of a blender, and then filtered through an autoclave muslin cloth. The aloe vera juice thus obtained was used for the testing of food preservative efficacy1–2 as novel preservatives by replacing sodium benzoate, methyl paraben and propyl paraben from the formula28.Ingredients: Zinc sulfate 40 gm, sulfurated potash 40 gm and purified water q.s. to 1,000 mL. Firstly, zinc sulfate and sulfurated potash were dissolved in 450 mL of water separately and filtered. Then, sulfurated potash solution was added to zinc sulfate with stirring. At last, the required amount of water was added and mixed thoroughly and sterilized. For preservative efficacy testing, the White lotion USP was prepared using the equimolar amount of compounds S. aureus MTCC 3,160, P. aeruginosa MTCC 1934, E. coli MTCC 45, C. albicans MTCC 183, and A. niger MTCC 282 were used as common contaminants in the study as prescribed in USP for preservative efficacy testing in the pharmaceutical preparations29.E. coli, P. aeruginosa, and S. aureus were incubated at the 37 °C for 24 h. The slants of C. albicans were incubated at 37 °C for 48 h, whereas; the slants of A. niger were incubated at 25 °C for 7 days29.The slants of .30. A concentration of 600 mg/kg or 600 ppm of sodium benzoate in aloe vera juice was taken as per food safety and standard guidelines. Equimolar quantity (0.0004 mol) of selected compound was taken as a preservative system in test samples. Challenged microbial cell suspension was inoculated the juice preparation with inoculum size never exceed more than 1%. After inoculation with microbes juice was incubated at room temperature for consecutive 28 days, and samples were collected on the 14th and 28th day of the experiment. The viable count of microorganisms was performed on nutrient agar (bacteria), and sabouraud dextrose agar (fungi) plates31. Each experiment was done in triplicate and log cfu/ml of juice was determined with comparison to standard.Preservative efficacy of the selected compound 1 and sodium benzoate (standard) was estimated in freshly prepared aloe vera juice as per the method with minor modifications as described by Ahlawat et al5–1 × 106 CFU/mL32. Inoculated samples were mixed thoroughly to ensure homogeneous microorganism distribution and incubated. The CFU/mL of the product was determined at an interval of 0 days, 7 days, 14 days, 21 days, and 28 days in agar plates. Log CFU/mL of white lotion USP was calculated as not as not less than 2.0 log reductions from initial count on 14th day of incubation and no increase in CFU from 14th day count to 28th day in case of bacteria and no increase from the initial calculated count on 14th day and 28th day in case of fungi33.White lotions USP was added in final containers and were used in the challenge test. The preparation was inoculated with a 0.5–1% volume of microbial inoculum having a concentration of 1 × 101 were stored at 40° ± 2 °C at 75% RH ± 5% RH (as per ICH guidelines) and were analyzed for the pH and cfu/ml of the product at 0, 1, 2, 3, 4, 5, and 6 months.Compound 1 was selected for stability study in their final container containing the formulation of Aole vera gel and White Lotion USP. Formulation having different preservatives i.e., standard and compound Data are expressed as mean values ± standard deviation or standard error as described in the legend of the figures and tables. Analysis of results was done by Kruskal–Wallis test as appropriate. Differences were considered to be statistically significant at P < 0.05. Statistical analyses were performed using MS excel data statistics analysis tool.34. Then the proposed library of amygdalin derivatives was again evaluated for their molecular docking behavior with the help of Schrodinger’s Maestro docking software35. Molecules were docked with the help of the G-6-P synthase crystallographic complex having pdb id 1moq. The Induced Fit docking (IFD) method of docking was utilized for the same. The predicted binding pattern revealed that synthesized ligand binds within the catalytic cavity of G-6-P synthase firmly via hydrogen bond formation, pi-pi stacking, and hydrophobic interactions. Two compounds 1 and 2, were selected via docking score, binding energy, and ADMET study parameters. Compound 1 showed the hydrogen bonding with Asp 354, Cys 300, and hydrophobic interactions with Val 605, Leu 601, while compound 2 showed hydrogen bonding with Asp 354, Ala 602, Ser 349, Ser 347, Thr 352, and hydrophobic interactions Leu 484, Cys 300. The synthesized compounds 1 and 2 possessed excellent dock score − 9.65, − 6.97, respectively and binding energy − 1.40 kJ/mol, − 51.31 kJ/mol, respectively as compared to standard drugs ciprofloxacin, ampicillin and fluconazole, dock score − 5.18, − 5.06, − 5.12, and binding energy − 37.16 kJ/mol, − 25.41 kJ/mol and − 23.15 kJ/mol, respectively. Hence, it is cleared that both the compounds 1 and 2 behave as G-6-P synthase inhibitor. Here, the inhibition of G-6-P synthase enzyme further evaluated by the outcomes of the inhibition likes antimicrobial activity. This further made the clearance behind the inhibition of G-6-P synthase enzyme by different proposed molecules. The molecular docking of the proposed amygdalin derivatives with the target site of G-6-P synthase (PDB ID 1MOQ) showed that all the selected compounds exhibited better binding affinity with different amino acid residues in active pocket of the enzyme. The results of molecular docking for different ligands within G-6-P synthase pocket and their interaction with different amino acid residues have been shown in Fig. The selection of amygdalin for further evaluation as preservative has been made on the basis of our prior evaluation in docking and absorption, distribution, metabolism and excretion (ADMET) study data36. All the synthesized compounds qualified the conditions for various descriptors like lipophilicity (LogP), hydrogen bond acceptor (HBA), hydrogen bond donor (HBD) and moleculat weight (MW). All parameters were in a suitable range for drug-like characteristics. In addition, according to Veber et al. (2002) for better bioavailability, rotatable bonds should be ≤ 10 as the rotatable bonds in ligand impart elasticity37. The values of QPlogBB should be > 1.0 CNS active compounds, and value < 1.0 CNS inactive compounds. QPPCaco cell permeability should be in a range from 4–7038. All the synthesized compounds exhibited a suitable drug-like profile and could be used for further evaluation as a novel preservative for food and pharmaceutical preparations.The compounds selected from docking have been further evaluated for their ADME parameters so that the selection of final preservative becomes easy. The evaluation of different ADMET parameters of selected amygdalin derivatives has been represented in Table (derivatives 1–2) by the reaction according to Vemula et al. (2006), outlined in Scheme 139. The chemical structural parts of all the synthesized compounds were confirmed by FTIR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analysis, which were in full agreement with their structures. The synthesis of amygdalin esters was completed by enzyme catalysis. In general, 40 ml of acetone containing 0.1 mol/L amygdalin and 0.3 mol/L vinyl ester was added in 1 g of Novozyme 435. The reaction mixtures were placed it in an incubator shaker at 200 rpm on 45 °C for 48 h. The filtration of reaction mixtures terminated the reaction.The amygdalin derivatives selected on the basis of docking and ADMET parameters were synthesized −1 (–OH) and appearance of a peak at 1,730 cm−1 and 1744 cm−1 (–C=O), respectively for compound 1 and compound 2. The change in chemical shift value, coupling constant and multiplicities were analyzed by 1HNMR and 13C NMR signals of synthesized compounds. The FTIR, 1H NMR and 13C NMR data confirmed the chemical structures of synthesized amygdalin derivatives. Final confirmation of the synthesized compounds was done by analyzing the mass spectrum of synthesized derivatives for molecular weight determination, and the Q-ToF Micro instrument was used as ion source. Most of the derivatives showed M+ (molecular ion peak), (M++1), (M++2) in positive chemical ionization, and (M1+), (M2+), M+ during negative chemical ionization mode. Finally, establishment of synthesis of amygdalin derivatives was done by elemental analysis where C, H, and N in percent were found within acceptable limits.Both of the compounds were synthesized according to the standard procedures as outlined in Scheme 1. The completion of the reaction was confirmed by TLC under UV lamp and FTIR. Formation of compounds 1 and 2 was further confirmed by peak shifted from 2,730 cm40. In the present study, DPPH radical scavenging assay method was utilized for the evaluation of the antioxidant profile of the synthesized compounds41. In this screening, compound 1 was observed as the most potent antioxidant compound  as compared to reference standard L-ascorbic acid . However, compound 2 showed moderate antioxidant activity . The antioxidant activity of the amygdalin was found 7.72 ± 0.03 µM42. Here, the better antioxidant property of amygdalin derivative shall be useful in the preservation of food, cosmetics, and pharmaceuticals43. All the results were expressed as mean ± standard deviation (n = 5) and results were found significant with Krukal-Wallis test (p < 0.05).The plant-based antioxidants can be used in food and pharmaceuticals to enhance their shelf life against oxidationK. reparati, P. mirabilis, P.aeruginosa, S. aureus, E. coli, C. albicans, and A. niger. Antimicrobial activity of the test compounds revealed that the compound 1 was found to be the most potent compound  as compared to the standard drugs streptomycin , ciprofloxacin , ampicillin  and fluconazole  using tube dilution method. Here, the results of MIC studies -(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl3- acrylate) showed antioxidant, antimicrobial, better preservative efficacy and prevent the change in pH as well microbial count of formulation for food as well as pharmaceutical products, which were in agreement with the results of molecular docking and highlight the mechanism of their preservative activity. Therefore, the synthesized amygdalin derivatives can be used as novel food and pharmaceutical preservatives to prevent them from microbial degradation.It has also been reported in our previous study that amygdalin can act as an active inhibitor of G-6-P synthase enzyme based upon the results of molecular docking and ADMET data


Fabp3 deletion antagonized the accumulation of phosphorylated αSyn, decrease in GABAergic neurons, and cognitive impairment caused by αSyn fibrils. Overall, the present study indicates that FABP3 mediates αSyn neurotoxicity in septal GABAergic neurons and the resultant cognitive impairment, and that FABP3 in this subpopulation could be a therapeutic target for dementia in synucleinopathies.In synucleinopathies, while motor symptoms are thought to be attributed to the accumulation of misfolded α-synuclein (αSyn) in nigral dopaminergic neurons, it remains to be elucidated how cognitive decline arises. Here, we investigated the effects of distinct αSyn strains on cognition and the related neuropathology in the medial septum/diagonal band (MS/DB), a key region for cognitive processing. Bilateral injection of αSyn fibrils into the dorsal striatum potently impaired cognition in mice. The cognitive decline was accompanied by accumulation of phosphorylated αSyn at Ser129 and reduction of gamma-aminobutyric acid (GABA)-ergic but not cholinergic neurons in the MS/DB. Since we have demonstrated that fatty acid-binding protein 3 (FABP3) is critical for αSyn neurotoxicity in nigral dopaminergic neurons, we investigated whether FABP3 also participates in αSyn pathology in the MS/DB and cognitive decline. FABP3 was highly expressed in GABAergic but rarely in cholinergic neurons in the MS/DB. Notably,  Synucleinopathies, including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), are neuropathologically characterized by Lewy bodies (LBs) and Lewy neurites (LNs), which contain α-synuclein (αSyn) in abundance . The mosThe medial septum/diagonal band (MS/DB) is one of the subcortical regions projecting glutamatergic, gamma-aminobutyric acid (GABA)-ergic, and cholinergic neurons to various areas, including the hippocampus and cerebral cortex ,10,11. TFabp3−/− mice, by treatment with a competitive inhibitor of LC-PUFAs binding to FABP3 [Long-chain polyunsaturated fatty acids (LC-PUFAs) have been documented to be a factor responsible for αSyn aggregation and oligomerization. While LC-PUFAs, including docosahexaenoic acid and arachidonic acid, are essential for brain development , they coto FABP3 ,35,36,37In the present study, we investigated the effects of αSyn strains on cognitive function, neuropathology in the MS/DB, and the relationship of FABP3. Intrastriatal injection of αSyn fibrils severely impaired cognition, and phosphorylated αSyn (pαSyn) at Ser129 accumulated in GABAergic but not in cholinergic neurons in the MS/DB. Notably, FABP3 was highly expressed in GABAergic neurons in the MS/DB, and its deletion antagonized αSyn fibril-induced cytotoxicity in GABAergic neurons and cognitive impairment. = 7.02, p = 0.002) and 60 days  = 18.17, p < 0.001). Intrastriatal injection of αSyn fibrils significantly reduced the latency to enter the dark compartment  = 24.12, p < 0.001), αSyn strain  = 8.40, p < 0.001), and day × αSyn strain  = 2.29, p = 0.039). On days 2 and 3 in the training sessions, αSyn fibrils significantly delayed the latency  = 5.95, p = 0.003). αSyn fibrils significantly impaired the retention of spatial memory  = 7.66, p = 0.004). Notably, αSyn fibrils significantly reduced the number of GAD67-positive cells  = 15.17, p = 0.001). Consistent with the reduction of GAD67-positive cells  = 3.58, p < 0.01; Expression levels of endogenous αSyn in the MS/DB should also be investigated as another factor that contributes to αSyn spreading and the toxicity. Additionally, it remains unclear whether FABP3, a critical molecule for αSyn neurotoxicity in the SNpc ,35,37, iFabp3−/− mice and evaluated pαSyn accumulation and decrease in GABAergic neurons. pαSyn accumulation in GAD67-positive cells was significantly reduced in Fabp3−/− mice compared to Fabp3+/+ counterparts (t(7.35) = 5.12, p < 0.01;  = 14.27, p = 0.001), αSyn strain  = 5.78, p = 0.026), and genotype × αSyn strain  = 7.51, p = 0.013). Decreased GAD67-positive cells seen in the MS/DB of αSyn fibril-injected Fabp3+/+ mice were restored in Fabp3−/− counterparts  = 6.48, p = 0.015; 60 days: F = 4.25, p = 0.047), αSyn strain  = 11.16, p < 0.001; 60 days: F = 9.21, p < 0.001), and genotype × αSyn strain  = 12.93, p < 0.001; 60 days: F = 10.58, p < 0.001). The shortened retention time seen in αSyn fibril-injected Fabp3+/+ mice was potently restored in Fabp3−/− counterparts  = 16.47, p < 0.001), genotype  = 17.91, p < 0.001), αSyn strain  = 7.73, p = 0.002), day × genotype  = 3.01, p = 0.045), day × αSyn strain  = 3.51, p = 0.007), genotype × αSyn strain  = 8.85, p < 0.001), day × genotype × αSyn strain  = 2.50, p = 0.039). Consistent with the results above  = 5.16, p = 0.011) and genotype × αSyn strain  = 4.79, p = 0.014) in terms of the latency to explore the target hole. Fabp3 deletion significantly improved the impaired retention of spatial memory seen in αSyn fibril-injected mice  Intrastriatal injection of αSyn fibrils leads to reduction of GABAergic neurons in the MS/DB and cognitive impairment. (2) FABP3 is highly expressed in GABAergic neurons in the MS/DB. (3) +) in PC12 cells [+-induced αSyn oligomerization and the resultant cell death when mutated FABP3 (F16S) that has little affinity for LC-PUFA binding is transfected in PC12 cells [We previously demonstrated that while FABP3 and αSyn make complexes and oligomerize in the SNpc of MPTP-treated mice, overexpression of the proteins fails to promote αSyn oligomerization in the absence of 1-methyl-4-phenylpyridinium , and intranigral injection of these patient-derived αSyn in mice induced nigrostriatal dopaminergic degeneration and motor impairments with relative severity in patients with MSA . AnotherSnca (A30P) compared to wild-type counterparts [As cholinergic neuronal loss in the MS/DB and the decreased levels in the cerebral cortex have been reported in DLB patients ,58, a chterparts ,70,71. TSnca-deficient mice injected with αSyn fibrils [Snca (A53T) in the locus coeruleus, which has projections with the septal nucleus [Fabp3 deletion, it is likely that FABP3 participates in αSyn pathology in not only nigral dopaminergic neurons but also other regions whose cell subpopulations highly express FABP3, such as septal GABAergic neurons. Further studies using region- or cellular subpopulation-specific knockdown of Fabp3 gene would warrant our hypothesis that the vulnerability to αSyn pathology in distinct cellular subpopulations is attributed to the expression levels of FABP3, in addition to those of endogenous αSyn and the extent of neural projection. In addition, αSyn levels of higher molecular weight and insoluble species should also be investigated in the MS/DB in this model and the relevance of FABP3 in order to further highlight the importance of αSyn pathology in the region for cognitive decline.Endogenous αSyn in naïve mice represented typical patterns of localization in the MS/DB, similar to other regions, as dense in presynaptic terminals and occasionally positive in cytoplasm. In addition, αSyn was not concentrated in either cholinergic or GABAergic neurons in the MS/DB. The spread of αSyn pathology was not observed in  fibrils , support fibrils ,44,45. C fibrils . Septal  nucleus ,75. Inje nucleus ,76,77. T nucleus ,47. Neve nucleus ,35,36,37 nucleus , we haveFabp3 deletion potently inhibited the pathology and cognitive impairments. These results highlighted the importance of FABP3 expression in αSyn pathology. Taken together, FABP3 could be a molecular target for both Parkinsonism and dementia in synucleinopathies.In conclusion, the present study documented that FABP3 partly contributes to αSyn propagation in GABAergic neurons in the MS/DB and cognitive impairments seen in mice injected intrastriatally with αSyn fibrils. αSyn pathology was well correlated with distinct localization of FABP3 in the MS/DB, and Fabp3+/+) male mice  were bred in a conventional environment with a 12/12-h light/dark cycle (light: 9:00–21:00) and freely accessible normal chow and water. Fabp3−/− male mice were generated on a C57BL/6 background [All animal experiments were approved by the Committee on Animal Experiments at Tohoku University  and conformed to the Regulations for Animal Experiments and Related Activities at Tohoku University. We made every effort to minimize the number of mice used and their discomfort. Wild-type C57BL/6 . Immunofluorescence was conducted as previously described [Each mouse was perfused and fixed with 4% paraformaldehyde solution, and the brain was post-fixed for 18–36 h at 4 °C. For the localization analyses of endogenous αSyn and FABP3, naïve escribed . PrimaryImages were acquired by a confocal microscope TCS SP8  and the z-stacks were reconstructed to 3D images with a software LAS X . The images of ChAT- or GAD67-positive cells in the entire MS/DB were captured with 10× or 20× objective lens, followed by tile-stitch processing using LAS X. The acquired images (three sections per mouse) were analyzed and counted in Fiji image J . For quantification of pαSyn, the images were captured with a 63× objective lens (six images from three sections per mouse). The anteroposterior coordinates from the bregma were 1.18 to 0.74 mm as described .Fabp3+/+ and Fabp3−/− male mice at the age of three months. Tissues were lysed in homogenizing buffer  with protease and phosphatase inhibitors and were centrifuged . The resultant supernatant was mixed with Laemmli’s buffer and boiled for 5 min. Proteins were separated in 13.5% SDS-PAGE and blotted on polyvinylidene difluoride membranes. Blots for αSyn were then crosslinked with 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min at room temperature [Protein extraction and immunoblotting analyses were conducted as previously described . MS/DB aperature . After bt-test for the results of the novel object recognition task , two-way (genotype × time or genotype × strain), or one-way ANOVA followed by post-hoc Tukey’s test. When repeated measures ANOVA was performed , followed by detection of F values by Greenhouse-Geisser ε correction. When ε values (estimating sphericity) > 0.1, corrected F values were adopted. Values of significant difference between groups were set as follows: * when p < 0.05, and ** when p < 0.01.The results are presented as box plots with whiskers delineating the 95th and 5th percentiles. The first and third quartiles are depicted by the bottom and top of the box, respectively. The median is represented by the line within the box. Statistical analyses were performed using JASP . Statistical significance was detected using two-tailed paired ion task A or two-


Ficus crocata(Miq.) Mart. ex Miq. (F. crocata) leaves (Ace-EFc) on cervical cancer cells, as well as its protective effect on hydrogen peroxide (H2O2)-induced lipoperoxidation and cytotoxicity in non-tumor HaCaT cells. Antioxidant activity was determined using the DPPH and ABTS radicals. Cell viability and lipoperoxidation were determined with MTT and 1-methyl-2-phenylindole assays, respectively. A model of H2O2-induced cytotoxicity and oxidative damage in HaCaT cells was established. HaCaT cells were exposed to the extract before or after exposure to H2O2, and oxidative damage and cell viability were evaluated. Ace-EFc inhibited the DPPH and ABTS radicals and showed a cytotoxic effect on SiHa and HeLa cells. Furthermore, the extract treatment had a protective effect on hydrogen peroxide-induced lipoperoxidation and cytotoxicity, avoiding the increase in MalonDiAldehyde (MDA) levels and the decrease in cell viability (p < 0.001). These results suggest that the metabolites of F. crocata leaves possess antioxidant and cytoprotective activity against oxidative damage. Thus, they could be useful for protecting cells from conditions that cause oxidative stress.Oxidative stress causes several chronic diseases including cancer. Some chemotherapeutic agents are not selective against tumor cells, causing oxidative stress in non-tumor cells. This study aimed to evaluate the cytotoxic effect of acetone extract of  After incubation for 16 h at room temperature, 1 mL of this solution was diluted in 60 mL of methanol. In both tests, methanol was employed as blank, while ascorbic acid (0.05–200 µg/mL) was used as standard antioxidant. The reactions between Ace-EFc and DPPH• or ABTS•+ were incubated in the dark at room temperature for 30 min, then read at 545 nm  and 734 nm , respectively. The half-maximal Inhibitory Concentration (IC50) was calculated through the linear equation (Y = mX + b) using GraphPad Prism v6.0 software. The percentage of radical inhibition was determined by the following formula:To evaluate the antiradical activity of Ace-EFc, 0.05–200 µg/mL of Ace-EFc was placed in 96-well plates , and 150 µL of 0.3 mM 2,2-DiOhenyl-1-PicrylHydrazyl . HaCaT, SiHa, and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium formula 12 (DMEM/F12) supplemented with 10% Fetal Bovine Serum (FBS), 1% antibiotic (Ampicillin/Streptomycin), and incubated at 37 °C in a 5% CO2 atmosphere at 95% humidity and were grown to 80% confluence. To establish H2O2 concentrations inducing cytotoxicity and oxidative damage, the HaCaT cells were exposed to 0, 200, 400, 600, 800, and 1000 µM of H2O2 during 0.5, 1, 2, and 4 h, and cell viability and lipoperoxidation were determined.HaCaT, SiHa, and HeLa cell lines were obtained and previously certified from 4 HaCaT, SiHa, or HeLa cells per well were cultured with DMEM/F12 medium with 10% FBS for 24 h. The cells were treated with 0–320 µg/mL of Ace-EFc for 24 h. After the treatment, the medium containing the extract was replaced by fresh basal medium and 100 µL of the MTT reagent was added for 4 h. The formazan crystals were diluted with Isopropanol, and the Optical Density (OD) of the supernatant was obtained at a wavelength of 545/630 nm . To assess the protective effect of Ace-EFc on H2O2-induced cytotoxicity, HaCaT cells were initially treated with Ace-EFc as described previously. The medium was discarded, and subsequently, cells were treated with H2O2 (1000 µM for 2h) according to the model established H2O2-induced cytotoxicity. The percentage of viable cells was determined by the following formula:Cell viability was evaluated using the MTT  cell proliferation colorimetric assay  according to the manufacturer’s instructions. Briefly, in a 96-well plate, 1 × 102O2 (1000 µM for 2h), according to the model established H2O2-induced cytotoxicity. The supernatant was recovered (2 mL), and the cells were trypsinized and resuspended in 65 µL PBS (pH 7.4), and vortexed for 1 min. To quantify MalonDiAldehyde (MDA) levels, 50 µL of the sample (supernatant or cells separately), 50 µL Milli Q water, 350 µL MPI (1-Methyl-2-OhenylIndole) reagent (10 mM), and 100 µL HCl (37%) were mixed, shaken, and they were incubated for 40 min at 45 °C. They were subsequently centrifuged at 4 °C at 7000 rpm for 10 min and the absorbance was read at 586 nm  [HaCaT cells were grown in a six-well plate to 80% confluence and were treated with 0–20 μg/mL of Ace-EFc for 24 h. Later, the medium was discarded, and subsequently, cells were treated with HMA, USA) . The datp < 0.05.Data analysis was performed using GraphPad Prism version 6.0 statistical software. The data were shown as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used with the Dunnett multiple comparison test. Statistically significant differences were considered when


C-glucosylation in PAINS membrane-interfering behavior, we disclose herein the first synthesis of 4-glucosylresveratrol, starting from 5-hydroxymethylbenzene-1,3-diol, via C-glucosylation, oxidation and Horner-Wadsworth-Emmons olefination, and resynthesize phloretin and genistein C-glucosides. We show that C-glucosylation generates compounds which are no longer able to modify membrane dipole potential. Therefore, it can be devised as a strategy to generate bioactive natural product derivatives that no longer act as membrane dipole potential modifiers. Our results offer a new technology towards rescuing bioactive polyphenols from their PAINS danger label through C–C ligation of sugars.The concept of Pan-Assay Interference Compounds (PAINS) is regarded as a threat to the recognition of the broad bioactivity of natural products. Based on the established relationship between altered membrane dipole potential and transmembrane protein conformation and function, we investigate here polyphenols' ability to induce changes in cell membrane dipole potential. Ultimately, we are interested in finding a tool to prevent polyphenol PAINS-type behavior and produce compounds less prone to untargeted and promiscuous interactions with the cell membrane. Di-8-ANEPPS fluorescence ratiometric measurements suggest that planar lipophilic polyphenols—phloretin, genistein and resveratrol—act by decreasing membrane dipole potential, especially in cholesterol-rich domains such as lipid rafts, which play a role in important cellular processes. These results provide a mechanism for their labelling as PAINS through their ability to disrupt cell membrane homeostasis. Aiming to explore the role of  Many of these compounds are natural products, among which genistein, resveratrol, epigallocatechin gallate (EGCG), quercetin or curcumin stand out for having been widely studied as potential therapeutic agents against a number of pathophysiological processes and conditions, including cancer4, inflammation5, diabetes6, and Alzheimer’s disease10. Confusing and contradictory results in clinical trials have been putting a question mark on the real therapeutic usefulness of these natural molecules16. However, while EGCG, quercetin and related compounds displaying catechol or hydroquinone groups are able to covalently inhibit protein activity upon autoxidation in enzyme inhibition assays (which might promptly justify their promiscuity)1, resveratrol and genistein lack such reactive moieties in their structure and thus ought to engage in much more subtle alterations in protein structure and conformation. In fact, being very planar and lipophilic molecules, they are believed to act as membrane-perturbing agents17. Nevertheless, both compounds have been tested in cell-free assays and found to be effective inhibitors of key enzymes such as BACE-118, DPP-419, PTP1B20, α-glucosidase21, 6-phosphofructo-1-kinase22, COX-123, PDEs24, among others. In this perspective, we reckon that new tools can be developed towards the conversion of these and other valuable scaffolds into safe and effective therapeutic lead molecules, with a reliable transposition of bioactivity from primary screening and pre-clinical assays to the final clinical stages, if the reason behind their odd behavior can be clarified and related to their PAINS-nature.PAINS were originally described by Baell & Holloway28. Henceforth, the described affinity of these compounds towards the so-called lipid rafts–which consist of more ordered domains, cholesterol and sphingomyelin (SM)-enriched, within the fluid bilayer enriched in unsaturated phosphatidylcholine (PC) 30–definitely calls for further investigation in model membrane systems contemplating membrane lateral heterogeneity. Indeed, lipid rafts are known to be related to membrane functionality by playing a crucial role in the regulation of membrane protein activity, protein and lipid trafficking, and, ultimately, signal transduction31. On the other hand, the membrane dipole potential is known to exert a crucial part in membrane permeability and ion transport, lipid-protein interactions, regulation of protein conformation and function, among other important roles35. Together with the transmembrane potential, the boundary potential is a key component of the electric profile associated with cell membranes, and can be subdivided into two subcomponents: the surface potential and the membrane dipole potential32. While surface potential is particularly related to the charges at the surface of the membrane36, membrane dipole potential results from the relative orientation between the electric dipoles of lipid headgroups and membrane-adsorbed water molecules37.Several authors have reported capacity of these types of compounds to modulate cell signaling pathways leading to desirable therapeutic outcomes in vivo38, we were encouraged to investigate if C-glucosylation of polyphenols exhibiting membrane-related PAINS behavior generates new structures devoid of this PAINS feature. Even though O-glucosylated compounds are much easier to synthesize, they are highly susceptible to hydrolysis in the gut, and therefore reach their targets as aglycones when administered orally–which should not occur with the chemically/enzymatically stable C-glucosyl derivatives. In this work, we tested this hypothesis using 1-palmitoyl-2--6-phenylhexa-1,3,5-trien-1-yl]phenyl}propanoyl)-sn-glycero-3-phosphocholine (DPH-PC) fluorescence anisotropy and (E)-3-(4-{2-[6-(dioctylamino)naphthalen-2-yl]vinyl}pyridin-1-ium-1-yl)propane-1-sulfonate (di-8-ANEPPS) fluorescence ratiometric measurements to evaluate different membrane physical parameters, namely the dipole potential, in large unilamellar vesicles (LUV) with diverse lipid compositions and phase behavior: (a) pure 1-palmitoyl-2-oleoyl-PC (POPC)–representative of the liquid disordered (ld) lipid phase; (b) POPC and cholesterol 1:1–as a model system for the liquid ordered (lo) phase; and (c) a ternary mixture of POPC, cholesterol and N-palmitoylsphingomyelin (PSM) 1:1:1–where the ld phase coexists with lo domains corresponding to lipid rafts39. All the lipid proportions indicated are molar ratios.On the basis of some of our previous work1)–which has been extensively used in membrane-interaction studies including dipole potential measurements35, genistein (2) and resveratrol (3)–two well-known PAINS with broad bioactivity and membrane-interfering behavior1.Three polyphenols were investigated Fig. : phloretC-glucosyl derivatives, nothofagin (4)38 and 8-glucosylgenistein 41, have been previously reported by our group and replicated for this study, we present herein the first synthesis of 4-glucosylresveratrol (E)-4-(β-d-glucopyranosyl)-5-(4-hydroxystyryl)benzene-1,3-diol, 6]. With this work, we ultimately aim to explore and rationalize the potential mechanisms behind the membrane-interfering behavior of PAINS, while elucidating the role of the sugar moiety in the impairment of such events.Whereas the synthesis of the first two C-glucosylation of this compound is very challenging and, in our experiments, proved to be unfruitful. In fact, several strategies were thoroughly explored before this goal could eventually be accomplished. One of the most robust approaches attempted was based on direct coupling of 2,3,4,6-tetra-O-benzyl-d-glucono-1,5-lactone to 2-bromoresveratrol. However, the anomeric C–C coupling of the gluconolactone and the brominated resveratrol in the presence of butyllithium at − 78 °C gave a complex mixture and did not lead to the desired product. We have then explored a new synthetic pathway, which culminated in the first synthesis of , presented in Scheme 7 and the pivaloyl-protected phenol 9. The regioisomers 10 and 11 were obtained in 43% global yield, and further purification afforded pure compound 10 as the major product, in 24% yield.Due to the highly conjugated nature of resveratrol, direct 12 in 50% yield over the three steps. A Horner-Wadsworth-Emmons olefination was then carried out with posphonate 13 in the presence of tert-BuOK in DMF to afford protected C-glucosyl resveratrol 15(E) isolated in 55% yield, which (E)-configuration is confirmed by the coupling constant of the olefinic proton at δ8.13 , ensuring that probe is not influencing membrane properties, particularly the membrane dipole potential, as reported by Clarke et al47. These authors also showed that probe-probe interaction, probe aggregation or membrane saturation are negligible for this probe concentration. In accordance with the previously described and well-known ability of cholesterol to increase membrane dipole potential51, POPC:cholesterol LUV (lo phase) presented significantly higher Rex values than pure POPC liposomes (ld phase), with a 1.5-fold increase, here displayed in Fig. ld and lo segregation in the ternary system, caused a decrease in this ratio to a value close to that observed for pure POPC LUV  in POPC:Chol and POPC:Chol:PSM-containing LUV than in liposomes solely containing POPC.In all lipid systems used herein, phloretin exerted the strongest decrease in the membrane dipole potential when compared to genistein and resveratrol. The observed decrease is in alignment with previous reports describing the dipole modifier-ability of phloretin and genistein17. Given the role of membrane dipole potential in protein conformation and function, particularly in cholesterol-containing domains such as lipid rafts60, our results indicate that a decrease in the membrane dipole potential may be one of the reasons behind this type of PAINS behavior.This reflects a more pronounced effect in cholesterol-rich domains, which was also observed in the case of genistein and resveratrol. Indeed, the ability of these and other PAINS to promiscuously alter membrane protein function has been described and attributed to cell membrane perturbations rather than specific protein binding35. The fact that the only hydroxy group of cholesterol is protected by the polar headgroups of phospholipids to avoid contact with water, like an umbrella60, may allow these polyphenols to lay at a more superficial location in the membrane. Indeed, it is possible that they end up placed in the membrane in such a way that their dipole moment is aligned with that of the membrane in an anti-parallel manner, causing the observed reduction in the overall membrane dipole potential. Resveratrol, for instance, has recently been shown to be distributed more at the surface of saturated 1,2-dipalmitoyl-PC (DPPC) monolayers, promptly establishing hydrogen bonds with the charged phosphate oxygen atoms of the phospholipid headgroups and/or carbonyl oxygen atoms of the acyl moieties61. This superficial positioning in the membrane may further lead to a remodelling effect through the rearrangement of existing hydrogen-bond networks between phospholipid headgroups and surface water molecules, which mediate membrane hydration mechanisms. In cholesterol-containing and more complex lipid systems, these interactions may even block the binding sites of the hydroxy group of cholesterol in phospholipids and sphingolipids62 leading to alterations in membrane thickness and fluidity63. In accordance, quercetin–a paradigmatic example of polyphenols with diverse and even promiscuous bioactivities–was recently shown to increase membrane hydration by interfering with cholesterol/sphingolipid-enriched domains, and is, like resveratrol, more superficially located in complex lipid mixtures64.The molecular mechanism underpinning this effect is probably connected to the lipophilic character and/or structural planarity of these types of molecules, combined with the multiple hydroxy groups present in both ends of their structure–which are able to act as hydrogen bond donor and/or acceptor groupsC-gluycosylated compounds show a completely different behavior. Our results clearly show that the observed dipole potential-decrease exerted by phloretin, genistein and resveratrol was fully abolished by the introduction of C-glucosyl moieties in each one of the three compounds  is actually a highly selective SGLT2 nanomolar inhibitor38 with antidiabetic, antithrombic and diuretic effects, being furthermore able to prevent high-glucose inflammation in vivo72. 8-Glucosylgenistein (5) is also a potent antidiabetic agent in vivo, with a strong inhibitory activity towards the formation of human islet amyloid polypeptides oligomers and fibrils41.At this point, it is important to highlight that 58 the changes in the membrane dipole potential (Δ ψd) induced by genistein, phloretin and phlorizin, measured by a non-fluorescence method in ternary 1,2-dioleoyl-PC (DOPC):Chol:SM (57:33:10 mol%) membranes were − 48 ± 10 mV, − 153 ± 18 mV and − 104 ± 5 mV, respectively. Thus, the effect of phloretin was quantitatively larger than that of phlorizin which was, in turn, larger than that of genistein. This is in full quantitative agreement with our measurements through di-8-ANEPPS ratiometric method.In the work by Chulkov et al.59 for different membrane compositions. Using the values of Δψd retrieved from these works and our Rex values, we found that there is a very good linear correlation between them (not shown). Therefore, our results can be used to quantitatively estimate dipole potential changes induced by the compounds. In addition, we have noticed that the variation in Rex previously obtained by us for the mixtures POPC:cholesterol and POPC:ergosterol with di-4-ANEPPS50, a probe very similar to di-8-ANEPPS is parallel to the trend in dipole potential reported for the same systems by Haldar et al.44, and the trend observed by Starke-Peterkovic et al.49 with 1,2-dimyristoyl-PC (DMPC):cholesterol upon increasing sterol concentration at 30 °C. Finally, the variation in Rex between POPC and POPC:cholesterol obtained by us with di-8-ANEPPS  lipid bilayers by fluorescence independent methods.These compounds were also studied by Efimova and OstroumovaRex and ψd given by Starke-Peterkovic T. et al. . This equation can be applied to di-8-ANEPPS ratiometric measurements when the excitation and emission wavelengths are the same as used in this work.Rescaling of our data was performed to match the dipole potential values reported by Haldar et al.n of Eq. . Insteadn of Eq.   planar lipid membranes, measured by Hidaka and Asami73 by dielectric spectroscopy. The value we obtained for phloretin is also in quantitative agreement with the studies by Gross et al.53 and Cseh and Benz74.The calculations led to the ΔψO-glucosyl derivative, genistin, is unable to change membrane dipole potential of ternary lipid mixtures (Δψd = − 1 mV)58. However, for phlorizin, only C-glucosylation affords a derivative completely lacking the ability to modify membrane dipole potential. Thus, our results suggest that C-glucosylation is a more powerful procedure to generate compounds counteracting the dipole potential modifying ability of polyphenols.Genistein is a weaker dipole modifying agent than phlorizin and its d in the order of—60 mV could cause a loss of ca. 70% of plasma membrane Na+/K+-ATPase activity45. A drop of the dipole potential induced by lyotropic anions, such as perchlorate, inhibited the conformational transition E1P(Na+)3 → E2P(Na+)3 of the enzyme in its phosphorylated state75. In the same work, it was shown that a change of electrical potential of no more − 16 mV would be sufficient to produce the decrease of affinity for ATP binding experimentally determined by the authors. Therefore, even considering possible shielding effects that could limit the effect of dipole modifying agents in a biological context, the values of Δψd for the aglycones and phlorizin in Table Considering our results in the ternary lipid mixture, which is closer to the composition of vertebrate cells plasma membrane outer leaflet, the decrease in dipole potential upon addition of polyphenols is 62 mV (genistein) or higher. Interestingly, a ΔψC-glucosides that could result from severe membrane disorganization or disruption, DPH-PC steady-state (SS) fluorescence anisotropy measurements in LUV were carried out in the same lipid systems used for dipole potential evaluation. The DPH group of DPH-PC is buried into the membrane, as expected from its hydrophobic character, and oriented in a parallel manner to the phospholipid fatty acid acyl chains76. This orientation allows the zwitterionic head of DPH-PC to be located at the membrane surface without the loss of the deep insertion of DPH. As shown by Kaiser et al.76, its average membrane location is closer to the bilayer center when compared to its analogue TMA-DPH and, in fact, deeper than DPH itself. It has been regarded as a relevant way of measuring lipid order in both artificial and biological membranes and was, therefore, chosen to probe the effects of each compound in our experiments78.To exclude other possible effects caused by the three polyphenol aglycones and their corresponding 79, the introduction of this sterol in the membrane significantly increases membrane order and packing, as manifested by the increase in DPH-PC SS fluorescence anisotropy in POPC:cholesterol liposomes when compared to pure POPC LUV, from 0.165 ± 0.002 to 0.247 ± 0.007 . Segregation of ld and lo phases in the ternary lipid system then led to a slight but statistically significant increase in membrane fluidity , as expected for an ld mole fraction of ca. 17%39 and an ld/lo partition coefficient of the probe close to 176.As expected from the previously described compacting effects of cholesterol81. Representative experimental decays of DPH-PC can be found in Fig. 83, are not markedly different for those membrane lipid compositions at 23 °C  were able to impact membrane packing in POPC liposomes (ld phase), as revealed by the decrease in DPH-PC SS fluorescence anisotropy when compared to controls 64. In addition, the polyphenol adopts a different orientation in the membrane when it is linked to a sugar88, which may also help to explain why 8-glucosylgenistein influences more membrane fluidity than its aglycone, genistein. On the other hand, the distinct location and orientation of the molecule may also explain the decreased ability of 8-glucosylgenistein to modify the membrane dipole potential. In the case of resveratrol and C-glucosyl resveratrol, both compounds have the most significant effect on membrane fluidity. However, the effect on membrane dipole potential is apparent only for the aglycone. Thus, even C-glucosylated compounds with the ability to increase membrane fluidity, and therefore unequivocally partitioning to the membrane phase, do not behave as dipole potential modifiers, in contrast to their aglycone counterpart.It could be possible that a smaller effect of the ity Fig. A, in somC-glucosyl derivatives, we assessed whether the interaction with complex membrane models would be altered in the presence of the sugar moiety. Notably, the first synthesis of 4-glucosylresveratrol (6) was accomplished for this purpose, and this expedient route is herein presented. Our DPH-PC SS fluorescence anisotropy results do not support the hypothesis that aglycones 1–3 owe their membrane-interfering behavior to the induction of changes in membrane order and compactness, at least at the level of the membrane hydrophobic core. However, we do have consistent results allowing to deduce that they are related to a membrane dipole modifying activity, as shown by di-8-ANEPPS fluorescence spectroscopy ratiometric measurements, with greater differences observed for cholesterol-containing membranes when compared to controls. These results indicate that PAINS may interfere with cholesterol-rich domains such as lipid rafts, probably by affecting the interactions at the membrane/water interface, with potential consequences in terms of the regulation of protein conformation and activity, lipid and protein sorting and trafficking, and signal transduction. Furthermore, all C-glucosides were able to fully prevent these changes in membrane dipole potential, as the membrane dipole potential observed in their presence did not differ from that of normal controls (LUV in the absence of any compound). We suggest that the sugar moiety alters the capacity to rearrange hydrogen-bond networks and hydration layer at the membrane surface, most likely due to the creation of multiple small electric dipoles by their hydroxy groups, which may therefore counteract the dramatic changes in the membrane dipole potential caused by the aglycones. This hypothesis is here exemplified for the pair genistein (2) / 8-glucosylgenistein (5) in Fig. We have studied a set of polyphenols with known PAINS-type behavior related to membrane perturbation or membrane dipole modifying activity. After synthesizing their 1), nothofagin (4) and 8-glucosylgenistein (5) were conducted according to previously described methodologies41. Genistein (2) and resveratrol (3) were purchased from Sigma Aldrich and TCI chemicals, respectively. All reactions were monitored by thin layer chromatography (TLC), which was carried out on aluminum sheets (20 × 20 cm) coated with silica gel 60 F-254, 0.2 mm thick (Merck) with detection by charring with 10% H2SO4 in ethanol. Column chromatography (CC) was performed using silica gel 230–400 mesh (Merck). Melting points were measured with a SMP3 melting point apparatus, Stuart Scientific, Bibby. Optical rotations were measured with a PerkinElmer 343 polarimeter. Nuclear Magnetic Resonance (NMR) experiments were recorded on a Bruker Avance 400 spectrometer at 298 K, operating at 100.62 MHz for 13C and at 400.13 MHz for 1H for solutions in CDCl3, (CD3)2CO or MeOD (Sigma-Aldrich). Chemical shifts are expressed in δ (ppm) and the proton coupling constants J in Hertz (Hz), and spectra were assigned using appropriate COSY, DEPT, HMQC, and HMBC spectra. Carbon labeling for NMR assignment was as follows: The olefinic carbons are labelled as C-a and C-b, ring A is labelled from C-1 to C-6, ring B from C-1′ to C-6′ and glucosyl carbons as C-1ʺ to C-6ʺ. The abbreviation Piv is used for the group COC(CH3)3 . High resolution mass spectra of new compounds were acquired on a Bruker Daltonics HR QqTOF Impact II mass spectrometer . The nebulizer gas (N2) pressure was set to 1.4 bar, and the drying gas (N2) flow rate was set to 4.0 L/minute at a temperature of 200 °C. The capillary voltage was set to 4500 V and the charging voltage was set to 2000 V.HPLC grade solvents and reagents were obtained from commercial suppliers and were used without further purification. The syntheses of phloretin  and DMAP  in pyridine (8 mL) at 0 °C. The mixture was stirred at room temperature. After 18 h another portion of pivaloyl chloride  was added and the mixture stirred for another 2 h. The crude was washed with HCl 2 M and extracted with DCM. The residue was dried with MgSO4 and the solvent evaporated under reduced pressure. The residue was purified by column chromatography (CyHex/EtOAc 10:1 → 6:1) to render compound 9 in 60% yield. Rf (CyHex/EtOAc 1:1) = 0.64; 1H NMR (CDCl3) δ (ppm) 6.38 , 6.30 , 4.97 , 1.20 . 13C NMR (CDCl3) δ (ppm) 179.6 (CO), 157.1 (C-3 and C-5), 139.1 (C-1), 107.2 (C-2 and C-6), 102.7 (C-4), 66.1 (CH2), 39.0 (OCOCq), 27.3 (CH3). HRMS-ESI (m/z): [M + H]+ calcd for C12H16O4 225.1121, found 225.1122. Pivaloyl chloride  was added in 3 portions to a solution of 5-(hydroxymethyl)benzene-1,3-diol 7  and compound 9  were dissolved in a 1:1 mixture of DCM/ACN (80 mL). Drierite (500 mg) was then added, and the reaction was kept at –40 °C under N2 atmosphere. TMSOTf  was added at low temperature and then the reaction stirred overnight at room temperature. After adding few drops of NEt3, the crude was washed with NaHCO3, extracted with DCM, washed with brine, dried over MgSO4 and concentrated in vacuum. The residue was purified by column chromatography (P.Ether/Acetone 10:1 → 7:1), affording a mixture of regioisomers 10 and 11 in 43% global yield. Two more column chromatography purification steps were necessary to isolate compounds 10 and 11 in 24% and 19% yield, respectively. Glycosyl donor f (P.Ether/Acetone 3:1) = 0.44; c 1, CHCl3); 1H NMR (CDCl3) δ 7.33–7.26 , 7.23–7.10 , 6.94 , 6.45 , 6.42 , 5.23, 5.20 , 4.97, 4.94 , 4.90–4.84 , 4.70 , 4.60, 4.57 , 4.52, 4.49 ; 4.45, 4.42 , 3.91 , 3.86–3.64 , 3.54 , 1.18 . 13C NMR (CDCl3) δ 178.4 (CO), 157.9 (C-3), 157.0 (C-5), 138.6, 137.9, 137.7, 137.4 (PhCq), 136.6 (C-1), 128.5–127.6 (PhCH), 113.8 (C-2), 108.4 (C-6), 104.9 (C-4), 86.2 (C-3ʹ), 81.4 (C-2ʹ), 78.6 (C-5ʹ), 77.1 , 75.7 (PhCH2), 75.4 (PhCH2), 75.3 (PhCH2-4′), 73.4 (PhCH2), 67.7 (C-6ʹ), 64.5 (CH2-OPiv), 38.8 [C(CH3)3], 27.2 (CH3). HRMS-ESI (m/z): [M + H]+ calcd for C46H51O9 747.3528, found 747.3528. Rf (P.Ether/Acetone 3:1) = 0.39; c 1, CHCl3). 1H NMR (CDCl3) δ 7.40–7.19 , 7.15 , 7.05–6.95 , 6.48 , 5.01 , 4.98 , 4.88 , 4.85, 4.82 , 4.69, 4.66 , 4.61, 4.58 , 4.55, 4.52 , 4.47, 4.44 , 4.24, 4.21 , 3.92 , 3.85–3.74 , 3.69, 3,67 , 3.58 , 1.24 . 13C NMR (CDCl3) δ 178.4 (CO), 155.6 , 138.7 (PhC´), 138.3 (PhC), 137.8, 137.6, 136.2 (PhC´), 128.8–127.5 (PhCH) 110.8 (C-4), 109.1 , 86.3 (C-3ʹ), 82.3 (C-2ʹ), 78.7 (C-5ʹ), 77.1 (C-4ʹ), 76.3 (PhCH2-2ʹ), 75.6 (C-1ʹ), 75.3 (PhCH2-4ʹ), 75.3 (PhCH2-4ʹ), 73.4 (PhCH2-6ʹ), 67.4 (C-6ʹ), 65.5 (CH2-OPiv), 38.8 [C(CH3)], 27.3 (CH3). HRMS-ESI (m/z): [M + H]+ calcd for C46H51O9 747.3528, found 747.3526. RC-glucosyl phenol 10  was dissolved in DMF (10 mL). To this solution, potassium carbonate  was added at 0 °C and stirred for 10 min at 0 °C. Then, benzyl bromide  was added and the reaction was stirred at room temperature for 1.5 h. After completion, the reaction was neutralized with HCl, 2 M, extracted with DCM, washed with brine, dried over MgSO4, and concentrated under vacuum. The benzylation product was isolated by column chromatography (P.Ether/Acetone 11:1 → 9:1) in 85% yield. Lithium hydroxide dissolved in a 1:1 mixture of MeOH/H2O (24 mL) was added to a solution of the C-glycosyl compound  in MeOH (10 mL). The mixture was refluxed for 36 h and then neutralized with Amberlite IR 120 H+. After filtration and evaporation of the solvent, purification was carried out by column chromatography (Hex/EtOAc 5:1 → 4:1) to render the hydrolysed product in 68% yield. Finally, to a suspension of pyridomium chlorochromate  in DCM (8 mL), a solution of the alcohol in DCM (8 mL) was added. The reaction was stirred at room temperature for 3 h and, after completion, the residue was washed with H2O and extracted with DCM. The product was purified by column chromatography (Hex/EtOAc 6:1 → 5:1) to afford compound 12 in 81% yield. Rf (Hex/EtOAc 5:1) = 0.35; c 1, CHCl3); 1H NMR (CDCl3) δ 10.76 , 7.47–7.06 , 6.88 , 6.74 , 5.25 , 5.09 , 4.96–4.81 , 4.62–4.42 , 4.02, 3,99 , 3.85–3.64 , 3.59 . 13C NMR (CDCl3) δ 192.2 (CO), 159.3 (C-5), 158.1 (C-3), 138.5, 138.06, 138.04, 137.7 (PhC), 137.5 (C-1), 136.4, 136.2, (PC), 128.6—127.0 (PhCH), 106.3 (C-4), 104.3 (C-6), 87.2 (C-3ʹ) , 79.6 (C-5ʹ), 78.2 , 76.3 (PhCH2-2ʹ), 75.6 (PhCH2-3ʹ), 74.5 (PhCH2), 73.6 (C-1ʹ), 73.4 (PhCH2-6ʹ), 71.1 (PhCH2-Phenol), 70.2 (PhCH2-phenol), 68.8 (C-6ʹ). HRMS-ESI (m/z): [M + H]+ calcd for C55H53O8 841.3735, found 841.3730.tert-butoxide was added to a solution of benzaldehyde 12  and phosphonate 13  in DMF at 0 °C. The mixture was warmed to room temperature and stirred for 2 h. After completion, the mixture was diluted with water, extracted several times with EtOAc, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (P.Ether/Acetone 22:1 → 20:1) to afford a mixture of isomers Z and E (1:2.3 ratio) in 60% global yield. Further purification steps afforded compound 15 in 55% yield. Rf (Hex/Acetone 7:1) = 0.52; 1H NMR (CDCl3) δ 8.13 , 7.46–7.16 , 6.98–6.89 , 6.85–6.75 , 6.54 , 5.28 , 5.12 , 5.06 , 5.02–4.82 , 4.74,4.71 ,4.68, 4.65 , 4.57, 4.54 , 4.43, 4.40 , 3.96–3.85 , 3.83–3.75 , 3.60 . 13C NMR (CDCl3) δ 159.3 (C-5), 158.6 (C-3), 158.4 (C-4ʹ), 140.5 (C-1), 138.7, 138.5, 138.4, 138.4 (PhCsugar), 137.2, 136.9 136.9 (PhC-resveratrol), 130.7 (C-1ʹ), 130.2 (C-b), 128.6—127.2 (37xPhCH), 126.0 (C-a), 117.8 (C-2), 115.1 (2xPhCH), 105.1 (C-6), 100.3 (C-4), 87.3 (C-3ʹ´), 81.9 (C-2ʹ´), 78.9 (C-5ʹ´), 77.7 (C-4ʹ´), 75.8 (PhCH2), 75.1 (PhCH2-4ʹ´), 74.2 (PhCH2-2ʹ´), 74.1 (C-1ʹ´), 73.7 (PhCH2-6ʹ´), 71.2 (PhCH2-phenol at C-3), 70.1, 70.0 , 68.9 (C-6ʹ´). HRMS-ESI (m/z): [M + H]+ calcd for C69H65O8 1021.4674, found 1021.4673. Potassium 15  in DCM, BCl3 was added in a dropwise manner at -78 °C in N2 atmosphere. After stirring for 2 h, MeOH was added and then the solvent eliminated under reduced pressure in a 25 °C bath. The residue was purified by reverse phase column chromatography (H2O/MeOH 9:1 → 7:3) to give compound 6 in 35% yield. Rf (DCM/MeOH 3:1) = 0.42; 1H NMR (MeOD) δ 7.38 , 6.83–6.73 , 6.57 , 6.26 , 3.94–3.85 , 3.81, 3.78 , 3.57–3.45 , 3.44–3.38 . 13C NMR (MeOD) δ 159.0, 158.8, 158.5 , 141.9 (C-1), 132.0 (C-5), 131.1 (C-b), 129.8 (C-a), 129.1 (C-2′ and C-6′), 116.6 (C-3′ and C-5′), 115.9 (C-2), 106.4 (C-6) 103.9 (C-4), 82.3 (C-5ʺ), 80.2 (C-2ʺ), 73.9 (C-1ʺ), 71.8, 71.4 , 62.8 (C-6ʺ). HRMS-ESI (m/z): [M + H]+ calcd for C20H23O8 391.1387, found 391.1389. To a solution of compound POPC and PSM were obtained from Avanti Polar Lipids. Di-8-ANEPPS was obtained from Biotium. DPH-PC was purchased from Molecular Probes. Cholesterol, minimum 99% and all other reagents, analytical grade were obtained from Sigma-Aldrich. Buffer solutions were prepared with ultrapure Milli-Q water at 18.2 MΩ.cm.93. Chol quantification was made by gravimetry. Probe concentration in stock solutions was determined spectrophotometrically.Phospholipid concentrations of POPC and PSM were determined gravimetrically and by inorganic phosphate quantification94. Briefly, the stock solution volume for the required final total lipid concentration was added to a vial, and the solvent evaporated with a mild, continuous flow of nitrogen, followed by overnight vacuum. Two identical samples were always prepared, with and without fluorophore, the latter to be used as blank. After hydration with buffer 10 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), pH 7.4, 150 mM NaCl, samples were submitted to vortex stirring and freeze-thaw cycles. LUV suspensions were formed by extrusion (Avanti Mini-extruder) at 60 °C, by forcing the multilamellar vesicle suspension 21 times through polycarbonate filters with 100 nm diameter pores  and left to reach equilibrium overnight. The probes were added from stock solutions in ethanol to an aliquot of freshly prepared LUVs and equilibrated overnight to ensure complete probe incorporation into the membrane95. The same volume of solvent (less than 1% v/v) was added the other aliquot of LUV suspension, to be used as blank.The interaction of plant polyphenols with lipid bilayers was studied by fluorescence spectroscopy in LUV suspensions, which were prepared according to previously described methods1—6 on membrane fluidity and dipole potential was studied in POPC, POPC:Chol (1:1) and POPC:Chol:PSM (1:1:1) lipid bilayers labelled with DPH-PC (in MeOH) or di-8-ANEPPS (in EtOH), respectively. Probe/lipid ratio was 1:500 in all experiments . Compounds were dissolved in DMSO and added to the LUV suspensions to achieve a concentration of 50 μM, with a final DMSO concentration of 2%, followed by an incubation period of 2 h at 23 °C prior to fluorescence measurements. Steady-state fluorescence anisotropy, ‹r›, was calculated according to the following equation:IXY represents the emission intensity reading with vertical (V) or horizontal (H) orientations of the excitation (X) and emission (Y) polarizers, and where G, obtained from the ratio of the intensities IHV/IHH, is a correction factor for the different detector sensitivity to vertical and horizontal polarized light. An adequate blank was subtracted from each intensity reading, and each set of four intensity components for each sample was measured seven times. Fluorescence anisotropy of DPH-PC was measured with λex at 369 nm and λem at 450 nm, with a 2 nm bandwidth. The membrane dipole potential was calculated from the fluorescence intensity ratio obtained for di-8-ANEPPS at 420 nm and 520 nm excitation wavelengths after background correction using a blank (unlabelled) sample. The blank signal was always below 0.7% of the respective labelled sample. The excitation spectra were obtained from 400 to 625 nm with emission at 635 nm. None of the compounds absorb light in the region of the excitation band of di-8-ANEPPS used. Emission spectra were recorded between 470 e 650 nm, at an excitation wavelength of 460 nm. The bandwidth was 5 nm in both excitation and emission.Fluorescence measurements were performed at 23 °C using a Horiba-Jobin Yvon Fluorolog 3–22 spectrofluorometer. The effect of compounds For time-resolved measurements by the single-photon-timing technique (SPT), nanoLED N-370 was used for the excitation of DPH-PC, and emission wavelength was 450 nm. The resolution of the detection system was 50 ps, and the number of counts in the peak channel was 10 000 − 20 000 for each sample. The time scale used for the analysis was 0.055517 ns/channel. The bandwidth was adjusted from the maximum value allowed by the instrumental setup (2.0–3.5 nm) to ensure an SPT regime. Data analysis was performed through a nonlinear least-squares iterative reconvolution method based on the Marquardt algorithm using the Time-Resolved Fluorescence Anisotropy Data Processor 1.4 program to obtain the fitting parameters. The fluorescence intensity decays were described by a sum of exponentials.Considering that to each component i of the decay corresponds a normalized pre-exponential factor (amplitude) The (intensity-weighted) mean fluorescence lifetime is then given byP < 0.05.Results are presented as the mean ± SD and each experiment was performed in triplicate. Statistical differences between compounds and control samples were assessed by one-way or two-way ANOVA followed by Tukey’s post-test performed using GraphPadPrism 6. Statistical differences were considered significant when Supplementary Information.