Bemisia tabaci is one of the most notorious agricultural pests in the world. A vicious circle among insect resistance, dose increased, environment and human body impaired as the overuse of synthetic pesticides are becoming increasingly evident. Entomopathogenic Beauveria sp. is known as an effective natural enemy to control B. tabaci. Therefore, this study aimed to purify and identify the biological compounds from Beauveria sp. LY2 via extensive chromatographic techniques, NMR and MS and evaluated for their insecticidal activities against B. tabaci via contact and feeding assay. The outcome identified that one new cerebroside, cerebroside F (1), nine known compounds, cerebroside B (2), bassiatin (3), methyl 1,4-dihydro-4-oxo-2-quinolinecarboxylate (4), cerevisterol (5), 9-hydroxycerevisterol (6), 6-dehydrocerevisterol (7), -ergosta-8(14),22-diene-3β,5α,6β,7α-tetrol (8), melithasterol B (9) and ergosterol peroxide (10) were isolated. Among the known compounds, methyl 1,4-dihydro-4-oxo- 2-quinolinecarboxylate (4) was isolated from natural origin for the first time. It is demonstrable from the results that compounds 3, 4 and 7 strongly featured insecticidal activities against B. tabaci, being the LC50 value as 10.59, 19.05, 26.59 μg/mL respectively in contact as well as 11.42, 5.66, 5.65 μg/mL respectively in feeding experiment. Moreover, no adverse effect on plant growth/height or phytotoxicity was observed on pepper, cucumber, tomato and cotton. The data from the current study has provided the foundation for the use of newly purified compounds against Bemisia tabaci as an alternative to synthetic chemical compounds. Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the world’s top 100 invasive species which can attack more than 600 agricultural plant-host species under field and greenhouse conditions2. Due to its piercing-sucking mouthpart, B. tabaci can directly cause plant weakness and indirectly transmit approximately 111 plant viruses3. Both nymphs and adults can also secrete honeydew, which can induce coal pollution. Besides B biotype (MEAM1) is the dominant species which significantly threatened many agricultural commodities in numerous countries6. Moreover, for the excessive use of synthetic pesticides have contributed to resistance development of B. tabaci. So far, 64 active ingredients have been reported for resistance from B. tabaci7.The whitefly, 8, and it can also result in the development of heritable resistance, pest resurgence and secondary pest problems. In contrast, bio-insecticides are safer for humans and more environmentally friendly. Moreover, plant and fungi are the sources of thousands of secondary metabolites9. Those secondary metabolites are ideal substitutes of those chemicals which play a significant role and possess a great potential on the field of biopesticides10.The injudicious and excessive use of broad-spectrum synthetic pesticide can do harm to human health and environmentBeauveria sp.  is a facultative entomopathogen with an extremely broad spectrum which is used as a commercial biopesticide against many agriculturally important insect pests11. The metabolites from Beauveria sp. can be divided into three main kinds, such as alkaloids , cyclodepsipetides  and benzoquinone (oosporein), many of them exert insecticidal13, anthelminthic14, synergistic antifungal15, antibacterial17, antiviral18 and cytotoxic activities19. However, research on the secondary metabolites of Beauveria sp. has a history of nearly 60 years, but the research on its activity is mainly focused on the field for medicinal use, and there are very few activities in the field of agriculture, in particular insecticidal activity. Although some studies have been conducted in isolation and identification of various compounds from natural resources, but, there is no research of bassiatin in the agricultural field per se. Therefore, this research not only supplements the gap in the insecticidal activity of bassiatin, but also adds two types of substances to the group of secondary metabolites of Beauveria sp., and evaluates their insecticidal effects in both feeding and contact toxicity effects.1–10 were obtained. Among them, compound 1 is a new cerebroside compound, 4 is a new natural product, and 2–10 are all confirmed to be known compounds after comparison with previous studies, they are cerebroside B (2)20, bassiatin (3)21, methyl 1,4-dihydro-4-oxo-2-quinolinecarboxylate (4)23, cerevisterol (5)24, 9-hydroxycerevisterol (6)25, 6-dehydrocerevisterol (7)26, -ergosta-8(14),22-diene-3β,5α,6β,7α-tetrol (8)27, melithasterol B (9)28, ergosterol peroxide (10)28. Compounds 1–2, 4–9 are all isolated from Beauveria sp. for the first time. The chemical structures of all the purified compounds (1–10) are presented in Fig. After silica gel column chromatography, gel chromatography and HPLC, compounds 1, white amorphous powder, possessed a molecular formula of C41H75NO10 by the positive HR-ESI-MS , requiring five indices of hydrogen deficiency. The 1H NMR spectrum , two mutually coupled trans olefinic protons at δH 5.74 , 5.50 , the diagnostic anomeric proton of β-glucopyranoside moiety at δH 4.26 , as well as two long-chain terminal methyl triplets at δH 0.89 . The 13C NMR spectrum , an amide acyl carbon at δC 177.2 , four olefinic carbon signals, a set of characteristic carbons corresponding to a β-D-glucopyranoside moiety appeared at δC 104.7 , 75.0 , 77.9 , 71.6 , 78.0  and 62.7 , other three oxygenated carbons at δC 73.0 (d), 72.7 (d) and 69.7 (t), as well as a series of long-chain aliphatic carbon signals. The above NMR features were similar to those of cerebroside B (2)20, also isolated from the current study, and the obvious difference between them only came from the olefinic methyl part of the main chain, in which the olefinic methyl signal was removed and replaced by a terminal olefinic methylene in 1, in addition, a keto carbonyl at δC 203.0 was newly detected. In the HMBC spectrum ] to the keto carbonyl carbon confirmed the presence of α, β-unsaturated carbonyl moiety. Furthermore, the correlations from H-7 [2.82 ] to C-5 [133.4 (d)] and C-8 [203.0 (s)] revealed the keto carbonyl at C-8. In view of their consistency of the chemical shifts and coupling constants, the configurations of C-2, C-3 and C-2′ were deduced to be the same as those of 2. The interpretation of the MS fragment ions . 1H NMR  δ 7.39 , 5.59 , 5.50 , 5.40 , 5.09 , 4.96 , 4.93 , 4.90 , 4.51 , 4.12 , 3.99 , 3.92 , 3.81 , 3.66 , 3.52 , 3.43 , 3.13 , 3.08 , 3.04 , 2.95 , 1.98 , 1.92 , 1.55 , 1.47–1.14 , 0.86 . 13C NMR  δ 174.14 (C-1′), 135.26 (C-9), 131.47 (C-5), 131.39 (C-4), 123.86 (C-8), 103.89 (C-1″), 77.28 (C-5″), 76.93 (C-3″), 73.78 (C-2″), 71.41 (C-2′), 70.93 (C-1), 70.39 (C-4″), 69.11 (C-3), 61.44 (C-6″), 53.22 (C-2), 39.43 (C-10) 34.86 (C-3′), 32.54 (C-6), 31.69–29.07 , 27.75 (C-7), 27.62 (C-11), 24.92 (C-4′), 22.49 (C-15′), 16.09 (C-19), 14.31 .Compound 3, colorless crystal, C15H19NO3, ESI–MS (m/z 261.00 [M]+) 1H NMR  δ 7.33 , 7.13 , 4.41 , 3.24 , 3.03 , 3.01 , 2.30 , 0.85 , 0.77 . 13C NMR  δ 167.38 , 165.64 , 134.26 , 129.89 , 129.34 , 128.33 , 81.38 , 62.87 , 37.19 , 32.53 , 29.77 , 18.68 , 15.24 .Compound 4, white amorphous powder, C11H9NO3, ESI-MS (m/z 202.20 [M-H]-). 1H NMR  δ 9.26 , 8.36 , 7.67 , 7.48 , 7.39 , 6.99 , 4.04 . 13C NMR  δ179.55 (C-4), 163.40 (C-9), 138.96 (C-1a), 136.10 (C-2), 133.10 (C-7), 126.34 (C-4a), 126.27 (C-5), 124.51 (C-6), 117.97 (C-8), 111.65 (C-3), 53.76 (C-10).Compound 5, white amorphous powder, C28H46O3, ESI–MS (m/z 431.20 [M + H]+). 1H NMR  δ 5.36 , 5.23 , 5.16 , 4.08 , 3.62 , 1.09 , 1.03 , 0.92 , 0.83 , 0.60 . 13C NMR  δ144.03 (C-8), 135.39 (C-22), 132.20 (C-23), 117.56 (C-7), 75.98 (C-5), 73.69 (C-6), 67.74 (C-3), 56.01 (C-17), 54.77 (C-14), 43.78 (C-13), 43.49 (C-9), 42.83 (C-24), 40.40 (C-20), 39.49 (C-4), 39.24 (C-12), 37.16 (C-20), 33.09 (C-25), 32.98 (C-1), 30.88 (C-2), 27.91 (C-16), 22.90 (C-15), 22.06 (C-11), 21.13 (C-21), 19.96 (C-26), 19.65 (C-27), 18.85 (C-19), 17.60 (C-28), 12.34 (C-18).Compound 6, white amorphous powder, C28H46O4, HR-ESI–MS (m/z 915.6686 [2 M + Na]+). 1H NMR  δ 5.36 , 5.26 , 4.03 , 3.68 , 2.53 , 1.15 , 1.07 , 0.98 , 0.89 , 0.69 . 13C NMR  δ 144.65 (C-8), 137.84 (C-22), 134.17 (C-23), 121.89 (C-7), 79.77 (C-5), 76.95 (C-9), 74.57 (C-6), 69.03 (C-3), 58.24 (C-17), 52.74 (C-14), 45.68 (C-13), 45.22 (C-24), 42.70 (C-20), 42.28 (C-10), 41.61 (C-4), 37.39 (C-12), 35.24 (C-25), 32.50 (C-2), 30.06 (C-11), 30.03(C-16), 29.12 (C-1), 24.87 (C-15), 23.09 (C-19), 22.50 (C-21), 21.33 (C-26), 20.95 (C-27), 19.07 (C-28), 13.13 (C-18).Compound 7, white amorphous powder, C28H44O3, ESI–MS (m/z 429.20 [M + H]+). 1H NMR  δ 5.65 , 5.24 , 5.16 , 4.03 , 2.52, 1.04 , 0.96 , 0.92 , 0.83 , 0.61 . 13C NMR  δ 198.31 (C-6), 165.32 (C-8), 135.02 (C-22), 132.51 (C-23), 119.71 (C-7), 77.81 (C-5), 67.50 (C-3), 56.05 (C-17), 55.82 (C-14), 44.78 (C-9), 43.90 (C-13), 42.82 (C-24), 40.46 (C-20), 40.28 (C-12), 38.85 (C-4), 36.51 (C-10), 33.06 (C-25), 30.37 (C-2), 30.23 (C-1), 29.71 (C-16), 27.86 (C-16), 22.49 (C-11), 21.96 (C-15), 21.11 (C-21), 19.95 (C-26), 19.65 (C-27), 17.58 (C-19), 16.43 (C-28), 12.70 (C-18).Compound 8, white amorphous powder, C28H46O4, ESI–MS (m/z 447.10 [M + H]+). 1H NMR  δ 5.20 , 4.42 , 3.91, 3.14 , 2.61 , 1.02 , 0.91 , 0.87 , 0.83 . 13C NMR  δ152.62 (C-14), 135.27 (C-22), 132.26 (C-23), 125.19 (C-8), 68.69 (C-3), 67.80 (C-5), 65.08 (C-6), 61.35 (C-7), 56.83 (C-17), 42.98 (C-24), 42.85 (C-13), 39.58 (C-20), 39.25 (C-3), 38.74 (C-9), 36.60 (C-12), 35.85 (C-10), 33.10 (C-26), 32.20 (C-1), 31.11 (C-2), 27.18 (C-16), 24.96 (C-15), 21.24 (C-21), 19.97 (C-28), 19.67 (C-27), 19.00 (C-11), 18.07 (C-19), 17.62 (C-18), 16.53 (C-25).Compound 9, white amorphous powder, C28H44O3, ESI–MS (m/z 451.30 [M + Na]+). 1H NMR  δ 5.19 , 4.22 , 3.95 , 3.31 , 1.14 , 1.02 , 0.91 , 0.83 , 0.59 . 13C NMR  δ 135.56 (C-22), 134.47 (C-9), 132.01 (C-23), 126.94 (C-8), 68.57 (C-3), 67.12 (C-7), 65.66 (C-5), 62.62 (C-6), 49.60 (C-14), 42.84 (C-24), 42.09 (C-13), 40.41 (C-20), 39.16 (C-4), 38.00 (C-10), 35.68 (C-12), 33.10(C-25), 30.84 (C-2), 30.20 (C-1), 29.01 (C-16), 23.85 (C-15), 23.4 1(C-11), 22.81 (C-19), 20.96 (C-21), 19.97 (C-27), 19.65 (C-26), 17.66 (C-28), 11.29 (C-18).Compound 10, white amorphous powder, C28H44O3, ESI–MS (m/z 429.20 [M + H]+). 1H NMR  δ 6.50 , 6.24 , 5.22 , 5.14 , 3.97 , 1.00 , 0.91 , 0.88 , 0.83 , 0.82 . 13C NMR  δ 135.44 , 135.22 , 132.32 , 130.76 , 82.17 , 79.44 , 66.46 , 56.20 , 51.69 , 51.09 , 44.57 , 42.78 , 39.74 , 39.35 , 36.97 , 36.93 , 34.70 , 33.07 , 30.11 , 28.65 , 23.40 , 20.88 , 20.63 , 19.95 , 19.65 , 18.18 , 17.56 , 12.87 .Compound 1–10 were evaluated for the insecticidal activity against B. tabaci. The results of the mean mortality via both contact and feeding toxicity was exhibited in Fig. 50 value at 72 h were presented in Table After the isolation, purification and identification, compounds 1, 41.67% mortality was reported at 72 h at 50 μg/mL via contact assay. Similarly, 38.33% mortality was displayed at the same concentration and time exposure via feeding assay  using a contact feeding method, that showed significant activity for isolated compounds30. The isolation of appropriate sterol from the Beauveria sp. and its activity against B. tabaci are in accordance with the findings of R.M. Zolotar et al. Similar results on insecticidal activity of ergosterol peroxide isolated from Nomuraea rileyi against tobacco cutworm were documented by Pannipa Prompiboon et al.31 who reported the moderate effects such as 46.7% mortality was caused against Spodoptera litura larvae via topical application at an interval of one week with 90–120 µg/insect, whereas, our research results showed 38.33% mortality at 72 h exposure at concentration of 50 μg/mL on B. tabaci. Additional biological activities of compounds ergosterol peroxide include antioxidant activities32, antimycobacterial activities33 inflammatory activities34. Furthermore, the research study did not find any published material with the same compounds that was isolated on insecticidal activity study. This suggests there is still great potential of prospects on finding new active molecules from known entomopathogenic fungi.The toxicity results of spinasterol,22,23-dihydrospinasterol reported by Maqsood et al. are in accordance with the results that documented LCB. tabaci is a potent agricultural pest with strong tendency to develop resistance against known pesticides, therefore new controlling agents are in constant need. In this study methyl 1,4-dihydro-4-oxo-2-quinolinecarboxylate (4), bassiatin (3), 6-dehydrocerevisterol (7) have shown promising insecticidal activity against B. tabaci. Compounds 1, 2, 4–9 are discovered in Beauveria sp. for the first time and compound 4 was discovered in natural products for the first time. Compound 4 was previously synthesized by Mazzoni22, even this is a known compound, we still use 2D NMR to fix the structure, due to no 13C NMR data was reported in the literature. However, our findings on phytotoxicity and growth parameter/ height displayed that no phytotocxic effects on plants leaves and plant heights were observed which showed the safety profile of the isolated compounds.B. tabaci is limited. The outcomes from this current study offered that isolated compounds from Beauveria sp. useful for the control of sucking pest especially B. tabaci. Hence, the introduction of these biologically active compounds could be an effective alternative and potential means to control such pests.Although different biological control approaches have been employed in current agriculture systems to control pest on crops and vegetables, however, the use of fungal based biological compounds against NMR spectra were captured on an Avance-600 NMR spectrometer  at room temperature. High-resolution electrospray ionization mass spectrometry (HRESIMS) spectra data were recorded on a 6500 series quadrupole-time-of-flight (Q-TOF) mass spectrometer . Mass spectrometry also recorded on LCMS 8050 . High-performance liquid chromatography (HPLC) analysis was performed on a 1260 Infinity LC system , and the column used was a 250 mm × 4.6 mm i.d., 5 µm, ZORBAX Eclipse XDB . Semipreparative HPLC was performed on a 1260 series system (Agilent), and the column used was a 250 mm × 9.4 mm i.d., 5 μm, ZORBAX Eclipse XDB (Agilent). Column chromatography was performed using silica gel (100−200 mesh)  and Sephadex LH-20 . All chemical reagents were purchased from a chemical reagent company  and used without further purification.Ostrinia furnacalis Guenée) cadaver which was collected from Liaoyang city , northeast of China in May, 2019. It was identified as Beauveria sp. LY2, according to morphological characters and molecular biological protocol by DNA amplification and sequencing of the ITS region35 . The fungal strain was prepared on sucrose agar with yeast extract slants and stored in Shenyang Agricultural University (SYAU), China, at 4 ℃.The fungal strain was isolated from an asian corn borner  was used. Put a 0.5 cm diameter fungi pancake with SDAY medium into a 250 mL Erlenmeyer flask which contains 40 mL of SDY medium, then incubated them at 25 ℃ with 180 rpm shaking speed for 3 days as to prepare the seed culture. In the second stage, Czapek–Dox medium  was used. 120 2L Erlenmeyer flasks, each contains 400 mL Czapek–Dox medium, were inoculated with 40 mL seed culture at 25 ℃ with 180 rpm shaking speed for 15 days. The fermentation cultures were centrifuged at 4 ℃ with 5000 rpm for 30 min to remove mycelia, then add 3% Amberlite XAD 16 resin into the broth at 25 ℃ with 180 rpm shaking speed for 4 h. Resin was collected by using 100 meshes gauze and extracted four times with methanol. The dried crude extract was harvested through reduced pressure concentration. The dried methanol extract was dissolved in 600 mL solution , the solution was extracted four times by 600 mL CH2Cl2. Collecting the resulted extract CH2Cl2 solution then concentrate it to produce 10.0 g solid brown residue.The 2Cl2-MeOH  as the mobile phase to afford six fractions, A to G. C was separated via silica gel chromatography with PE:EA  as the mobile phase to yield compound 3 (110 mg), compound 9 (7.3 mg) and compound 10 (14.4 mg). Fraction E was subjected to gel chromatography on Sephadex LH-20 eluted with CH2Cl2-MeOH (1:1) to obtain compound 5 (16.7 mg). D, F, G was purified by reverse-phase semi-preparative HPLC applying a MeOH-H2O gradient (contain 0.1% HCOOH) respectively. Compound 6 (2 mg) was gained from D with 90% MeOH on 40 min  and compound 8 (4.2 mg) were also obtained from D with 85% MeOH on 18 min and 20 min  was yielded from F with 77% MeOH on 28 min  and 2 (25.9 mg) was obtained from G with conditions with 98% MeOH on 10 min and 16 min respectively  are from Liaoning Key Laboratory of Economic and Applied Entomology was cultured in a controlled greenhouse at 25-27 ℃, 60%-70% relative humidity (RH) with 16:8 (light: dark) photoperiod, on around 2 months old cotton plants, Gossypium hirsutum.The whiteflies B-biotype 36. All items had to be completely sterile.One side of a lightproof bi-pass glass tube (3 cm inside diameter and 6 cm height) was covered with a stretched Parafilm M membrane (the first layer). Another side sealed with one piece of 100 meshes gauze and a rubber bandG. hirsutum into the container. Test containers were placed in a controlled greenhouse at 25–27 ℃, 60–70% under 16:8 (light:dark) photoperiod and mortality was recorded at 24, 48 and 72 h. Each iteration of the experiment consisted of four replicates for each concentration of each compound (4 replicates × 4 concentrations × 3 repetitions × 30 whiteflies per tube = 1440 B. tabaci per test compound).The whitefly diet comprised of 30% (w/v) sucrose with serial dilutions of each compound , 2.5 μg /mL acetamiprid was used as positive control, each diet contained 0.5% Tween 80 and 0.002% DMSO. Diet aliquots (200 μL) containing compounds were dispensed on top of the first layer of stretched Parafilm membrane over each container; another stretched Parafilm membrane was placed over the diet aliquots to prevent evaporation. 30 adults (2–5 d old) were aspirated from G. hirsutum into the container. Test containers were placed in the controlled greenhouse at 25–27 ℃, 60–70% under 16:8 (light: dark) photoperiod. B. tabaci mortality was recorded at 24, 48 and 72 h. Each iteration of the experiment consisted of four replicates for each concentration of the each compound .Used mixed solvent (methanol: dichloromethane = 1:1) to diluent each compound into serial concentrations , used 2.5 μg/mL acetamiprid as positive control, mixed solvent with compounds aliquots were dispensed into the container tube, rotated the tube once to let the solvent equally distributed and evaporate. 30 adults (2–5 d old) were aspirated from Capsicum annuum L., cultivar ‘Wanhao A5’ from SYAU, China), cucumber , Tomato  and cotton  were chosen as experimental objects and planted in nutrition pots from seeds for one month until they reached to specific leaves stage  in greenhouse with the temperature scale between 10 and 35 ℃. The equivalent crop plants with similar heights and proper leave sizes were picked and transplanted into 7 cm3 black plastic pots, then divided the crop plants into 8 groups, each group consists of 3 pots for each kind of crop plants.pepper  every 7 days for 2 weeks according to Chinese National Standard of Laboratory Test For Crop Safety Evaluation37 and data was calculated.The insecticidal compounds All the studies on plants totally complied with relevant institutional, national, and international guidelines and legislation.All the calculated data on mortality and plant heights were analyzed via one way analysis of variance (ANOVA), the mean difference between treatments was envisioned for significance test by Duncan multiple range test DMRT with IBM-SPSS statistics 25.0 version software. Probit analysis was performed using EPA Probit analysis program version 1.5. 0.All the chemical structures including bold bonds and arrows were drawn with ChemDraw 19.0. The Fig. Beauveria sp. possess potential botanical agents. Results also demonstrated that B. tabaci produced significant sensitivity to isolated bassiatin (3) via both feeding and contact bioassay as well as methyl 1,4-dihydro-4-oxo-2-quinolinecarboxylate (4) and 6-dehydrocerevisterol (7) via feeding bioassay. cerevisterol (5), -ergosta-8(14),22-diene-3β,5α,6β,7α-tetrol (8), melithasterol B (9) and ergosterol peroxide (10) displayed moderate toxicity against this pest. In contrast, cerebrosides F (1) and cerebrosides B (2) showed much lower mortality than other compounds. Moreover, all the compounds were found safe without affecting the plants growth with no phytotoxicity. Therefore, except compounds 1 and 2, these compounds were introduced as alternatives to synthetic chemical insecticides. However, more research on the purification and characterization of bioactive compounds from entomopathogenic fungi is needed to be compared against B. tabaci and other agricultural insect pests in potential future research studies.The present research study indicated that the secondary metabolites from Supplementary Information.


R,5S)-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one, available by an efficient catalytic pyrolysis of cellulose, has been applied as a chiral building block in the synthesis of seven new nucleoside analogues, with structural modifications on the nucleobase moiety and on the carboxyl- derived unit. The inverted configuration by Mitsunobu reaction used in their synthesis was verified by 2D-NOESY correlations, supported by the optimized structure employing the DFT methods. An in silico screening of these compounds as inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase has been carried out in comparison with both remdesivir, a mono-phosphoramidate prodrug recently approved for COVID-19 treatment, and its ribonucleoside metabolite GS-441524. Drug-likeness prediction and data by docking calculation indicated compound 6 [=-methyl 5-(hydroxymethyl)-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrofuran-3-carboxylate] as the best candidate. Furthermore, molecular dynamics simulation showed a stable interaction of structure 6 in RNA-dependent RNA polymerase (RdRp) complex and a lower average atomic fluctuation than GS-441524, suggesting a well accommodation in the RdRp binding pocket.(1 Cellulose is the most biosynthesized organic substance on earth and is an abundant component of plants. According to the biorefinery concept, it represents a key biomass to produce fuels and chemicals. In line with the sustainability of bioresources, the conversion of the carbohydrate content in lignocellulosic waste represents an interesting valorization. Biomass-derived compounds are often rich in functionalization and stereocenters, so that to be crucial chiral pools for the synthesis of non-racemic molecules. 1) -1-hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one) , where x represents the energy of current structure, µ and σ are the average value and the standard deviation of energy in the “gold standard” protein population. For all structures, Z resulted about −1.35, meaning that each of them was satisfactory . The radFor evaluating the thermodynamic stability of the system, Root Mean Square Deviation (RMSD) was calculated, both for Cα and backbone as well as for all the heavy atoms. Similar values of the protein in the starting and in complexed structures were obtained, in the range of 1.2–1.9 Å . In gene6 and GS-441524, respectively  at 1 cm−1 resolution in the absorption region 4000–600 cm–1. A thin solid layer is obtained by evaporation of methanol solution of the sample. The instrument was purged with a constant dry air flux and clean ATR crystal as background was used. Spectra processing was made using Opus software package. NMR spectra were recorded on a Bruker-Avance 400 spectrometer using a 5-mm BBI probe 1H at 400 MHz and 13C at 100 MHz with values relative to TMS, in CDCl3 (δH 7.25 and δC 77.00 ppm) or acetone-d6 (δH 2.05 and δC 29 ppm) with δ values in ppm and J values in Hz; assignments were supported by polarization transfer (DEPT), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments; nuclear Overhauser enhancement (nOe) data from bidimensional NOESY experiments. Electron impact (EI)–MS and high-resolution HR-EI-MS spectra  were recorded with a Kratos MS80 mass spectrometer equipped with home-built computerized acquisition software. Electrospray ionization (ESI)-MS mass spectra were recorded using a Bruker Esquire-LC spectrometer by direct infusion of a methanol solution . High-resolution ESI-MS measurements were obtained by direct infusion using an Orbitrap Fusion Tribrid mass spectrometer.All reagents were purchased from Sigma Aldrich  and used without further purification. Preparation of amino acid rocedure . Microwa2).-Methyl 3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-carboxylate .-Methyl 5-(((tert-butyldimethylsilyl)oxy)methyl)-3-hydroxytetrahydrofuran-3-carboxylate  in dichloromethane (20 mL) was stirred with tert-butylchlorodimethylsilane  and imidazole  at room temperature for 2 h, monitoring the total conversion by TLC. The reaction mixture was concentrated in vacuo and the residue subjected to FC on silica gel, using hexane/ethyl acetate gradient elution as eluent, obtaining pure 3 as a colorless oil. TLC (hexane/EtOAc = 40:60 v/v): Rf = 0.88. Yield: 78%. FT-IR (cm−1): 2952 (w), 1727 (s), 1463 (w), 1238 (s), 1099 (s), 837 (vs), 779 (s). 1H-NMR  δ 4.25 , 4.13  3.79 , 3.78 , 3.73 , 3.67 , 2.30 , 2.02 , 0.85 , 0.03 .13C-NMR  δ 181.3 (COO), 80.9, 79.7, 77.5 (C1), 67.9 (C5), 57.9 (OMe), 37.5 (C3), 28.3 (tBu), 1.5 (SiMe2). EI-MS: m/z 290 , 233 (24), 159 (8), 73 (100).4).-Methyl 5-(((tert-butyldimethylsilyl)oxy)methyl)-3-(6-chloro-9H-purin-9-yl)tetrahydrofuran-3-carboxylate  was slowly added to triphenylphosphine  in THF (8 mL) under N2 atmosphere at 0 °C. After stirring for 30 min, 6-chloropurine  and compound 3  in THF (8 mL) were added and the reaction mixture refluxed for 24 h. The solvent was evaporated and the crude residue was purified by column chromatography using hexane/EtOAc gradient elution to recover 230 mg of 3 and pure 4 (252 mg) as a light yellow oil TLC (hexane/EtOAc = 70:30 v/v): Rf = 0.20. Yield 48%. [α]D20= +20.0 . FT-IR (cm−1): 2954 (m), 2930 (m), 2858 (w), 1747 (s), 1589 (s), 1562 (s), 1486 (m), 1472 (m), 1437 (m), 1400 (m), 1343 (m), 1297 (w), 1257 (s), 1220 (s), 1149 (s), 1092 (m), 1006 (m), 936 (m), 837 (s), 779 (m). 1H NMR  δ 8.69 , 8.27 , 4.73  4.47 , 4.37 , 3.71 , 3.69 , 3.60 , 3.16 , 2.69 , 0.74 , −0.07 . 13C-NMR  δ 169.5 (COO), 152.0 (C6′), 151.6, 143.5 (C2′), 131.9, 80.2 (C4), 74.7(C1), 70.3, 63.7 (C5), 53.8 (OMe), 37.5 (C3), 25.1 (tBu), −6.1 (SiMe2). ESI(+)-MS: m/z 449 [M+Na]+, 427 [M+H]+. HREI-MS: m/z 411.12448 ± 0.0010, , m/z 369.07799 ± 0.0010, .5).-Methyl 3-(6-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carboxylate  and tetrabutylammonium fluoride  were stirred for 2 h at room temperature. The crude product obtained by evaporation of the solvent was purified by FC using hexane/ethyl acetate gradient elution, to give pure 5. TLC (dichloromethane/MeOH = 90:10): Rf 0.18. Yield: 87%. HPLC analysis  under UV detection at 254 nm showed a single peak at Rt = 18.2 min. [α]D20 = +18.2 . FT-IR: 3400 , 2900 (w broad), 1742 (s), 1590 (s), 1563 (s), 1436 (m), 1398 (m), 1343 (m), 1220 (s), 1050 (m), 934 (m), 834 (m), 636 (m). 1H NMR  δ 8.65 , 8.40 , 4.78  4.46 , 4.39 , 3.68 , 3.81 , 3.51 , 3.10 , 2.76 . 13C NMR  δ 169.5 (COO), 151.9, 151.8 (C6′), 151.1, 144.2 (C2′), 132.0, 80.2 (C4), 74.5 (C2), 70.3 (C1), 62.7 (C5), 53.8 (OMe), 37.5 (C3). ESI(+)-MS: m/z 336 [M+Na]+, 313 [M+H]+. HRESI(+)-MS: m/z 313.07031 ± 0.0005, calcd. for C12H1435ClN4O4: 313.06981.6).-Methyl 5-(hydroxymethyl)-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrofuran-3-carboxylate  and N-methyl piperazine  in MeOH (1 mL) were microwave irradiated for 1.5 h at 70 °C. The crude mixture was purified by preparative TLC, using dichlorometane/MeOH = 85:15 v/v added of few drops of trimethylamine. TLC: (dichloromethane/MeOH = 80:20 v/v): Rf 0.63. Yield: 75%. HPLC analysis  under UV detection at 254 nm showed a single peak at Rt = 26.2 min. [α]D20 = +21.2 (6.66 mg/mL in MeOH). FT-IR: 3351 , 2923 , 1742 (m), 1693 (w), 1586 (s), 1453 (m), 1250 (m), 1046 (m), 793 (m), 646 (m). 1H NMR  δ 8.27 , 7.90 , 4.70  4.53 , 4.39 , 4.31 , 3.70 , 3.80 , 3.52 , 3.09 , 2.67 , 2.54 , 2.34 . 13C NMR  δ 170.2 (COO), 153.7, 152.3 (C6′), 151.2, 149.5, 136.5 (C2′), 79.9, 69.5, 63.2, 54.7, 53.4, 45.8, 45.6, 37.4 (C3). ESI(+)-MS: m/z 399 [M+Na]+, 377 [M+H]+; MS/MS (377): m/z 320, 162. HRESI(+)-MS: m/z 377.19344 ± 0.0003, [M+H+], calcd. for C17H25N6O4: 377.19318).7).-Methyl 5-(hydroxymethyl)-3-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-3-carboxylate  and methylamine  in MeOH (0.5 mL) were microwave irradiated for 20 min at 70 °C. The crude mixture was purified by preparative TLC was purified by preparative TLC, using dichloromethane/MeOH = 90:10 v/v added of few drops of trimethylamine, obtaining pure 7 as a light-yellow solid. TLC (dichloromethane/MeOH = 90:10 v/v). Rf 0.25. Yield 92%. [α]D20= +16.9 (12.5 mg/mL in MeOH). FT-IR (cm−1): 3364 broad w, 2925 m, 1740 m, 1623 s, 1456 w, 1376 w, 1297 w, 1241 m, 1045 m, 963 w, 645 s broad. 1H NMR  δ 8.25 , 7.97 , 6.28 , 4.85  4.43 , 4.37 , 3.65 , 3.83 , 3.51 , 3.26 , 3.05 , 2.98 , 2.83 , 3.05 . 13C NMR  δ 170.3 (COO), 155.1, 153.2 (C6′), 148.8, 138.4 (C2′), 119.7, 80.5, 74.5, 69.8 (C1), 62.6 (C5), 53.4 (OMe), 37.2 (C3), 27.2 (NMe). ESI(+)-MS: m/z 308 [M+H]+, MS/MS (308): m/z 290, 248; HRESI(+)-MS: m/z 308.13566 ± 0.0004, [M+H]+, calcd. for C13H18N5O4: 308.13533.8).-Isopropyl 3-(6-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carboxylate  and triazabicyclodecene  in i-PrOH (1 mL) were microwave irradiated for 30 min at 50 °C. The crude mixture was purified using CN-column chromatography using dichloromethane/MeOH gradient elution to obtain pure 8 as light-yellow solid. TLC: CN phase, dichloromethane/MeOH 99:1 v/v. Rf = 0.56. Yield 53%. [α]D20 = +26.1 (17.4 mg/mL in MeOH). FT-IR (cm−1): 3400 , 2925 (m), 1720 (s), 1688 (s), 1589 (m), 1453 (m), 1376 (m), 1228 (s), 1100 (s), 1039 (s), 827 (m), 603 (s). 1H NMR  δ 8.68 , 8.26 , 4.76  4.42 , 5.00 , 4.37 , 3.80 , 3.50 , 3.07 , 2.72 , 2.97 , 1.06 . 13C NMR  δ 168.2 (COO), 151.9, 151.0, 151.6 (C6′), 144.1 (C2′), 131.8, 80.2 (C4), 74.4 (C2), 71.2 (C1), 70.6 (i-PrO), 62.7 (C5), 37.4 (C3), 21.4 (i-PrO). ESI(+)-MS: m/z 341 [M+H]+, 323 [M+H-H2O]+. HRESI(+)-MS: m/z 341.10181 ± 0.0007, calcd. for C14H1835ClN4O4: 341.10111.9).-Methyl 3-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carboxylate  was slowly added to triphenylphosphine  under N2 atmosphere at 0 °C. After 30 min, 3  and adenine in THF were added, stirring under reflux for 10 h, monitoring by TLC (hexane/EtOAc 6:4 v/v with few drops of trimethylamine). The reaction mixture was concentrated and the residue subjected to FC using hexane/AcOEt added of 1% triethylamine gradient elution. The recovered product was then treated with TBAF in THF stirring 2 h at room temperature as described in the procedure for obtaining 5. The deprotected product was purified using CN-column chromatography with hexane/AcOEt gradient elution obtaining pure 9 as light yellow solid TLC: dichloromethane/MeOH 85:15 v/v. Rf = 0.32. Yield 52%. [α]D20 = +16.8 (8.57 mg/mL in MeOH). FT-IR (cm−1) 3193 , 2928 (m), 1704 (m), 1642 (m), 1581 (s), 1437 (s), 1250 (s), 1119 (vs), 837 (m). 1H NMR  δ 8.19 , 8.03 , 6.06 , 4.84  4.45 , 4.41 , 3.69 , 3.87 , 3.53 , 3.05 , 2.84 . 13C NMR  δ 170.0 (COO), 155.3, 152.7 (C6′), 150.0, 139.0 (C2′), 119.6, 80.4, 74.6, 69.8 (C1), 62.6 (C5), 53.5 (OMe), 37.3 (C3). EI-MS: m/z 293 [M+.]; HRESI(+)-MS: m/z 294.11985 ± 0.0003, [M+H] +, calcd. for C12H15N5O4: 294.11968.10).-Isopropyl 3-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carboxylate  and TBD  i-PrOH (1 mL) were microwave irradiated for 30 min at 50 °C. The crude product was purified by FC using dichloromethane/MeOH gradient elution, to obtain 10 as a pure white solid. TLC: dichloromethane/MeOH 90:10 v/v, +1% triethylamine. Rf = 0.57. Yield: 51%. [α]D20 = +13.0 (9.4 mg/mL in MeOH). FT-IR (cm−1) 3333 , 2926 (m), 2360 (w), 1734 (m), 1641 (s), 1600 (s), 1473 (m), 1292 (m), 1226 (s), 1102 (s), 1042 (m), 799 (m), 647 (m). 1H NMR  δ 8.21 , 8.03 , 5.93 , 4.81  4.45 , 5.03 , 4.40 , 3.87 , 3.54 , 3.04 , 2.80 , 1.08 . 13C NMR  δ 168.9 (COO), 155.3, 152.6, 150.1, 139.0, 119.5, 80.4, 74.5, 70.7, 70.0, 62.7, 37.1, 21.3. ESI(+)-MS: m/z 344 [M+Na]+, 322 [M+H]+; HRESI(+)-MS: m/z 322.15094 ± 0.0003, [M+H]+, calcd. for C14H20N5O4: 322.15098.11).-3-(6-Amino-9H-purin-9-yl)-5-(hydroxymethyl)-N-methyltetrahydrofuran-3-carboxamide  was slowly added to a solution of 9  in MeOH (1 mL) at 0 °C. The reaction was stirred overnight at r.t., then concentrated in vacuo and the residue subjected to FC, using dichloromethane/MeOH gradient elution, to obtain pure 11 as white solid. TLC: dichloromethane/MeOH 85:15 v/v. Rf: 0.23. Yield: 60%. [α]D20= +21.4 (7.8 g/mL in MeOH). FT-IR (cm−1): 3326 m broad, 2926 w, 1639 s, 1601 m, 1573 m, 1475 m, 1414 m, 1376 m, 1339 m, 1260 m, 1226 m, 1042 m, 644 s broad. 1H NMR  δ 8.16 , 8.14 , 6.65 , 4.65  4.56 , 4.29 , 3.50 , 3.46 , 3.14 , 2.67 , 2.66 . 13C NMR  δ 169.4 (COO), 156.2, 152.5 (C6′), 150.2, 139.6 (C2′), 120.1, 79.8, 73.9, 70.3 (C1), 63.3 (C5), 37.5 (C3), 25.7 (NMe). ESI(+)-MS m/z 293 [M+H]+; MS/MS(293): m/z 234; HRESI(+)-MS: m/z 293.13586±0.0003, [M+H]+, calcd. for C12H17N6O3: 293.13566.ADME prediction were performed using the Online Server Swiss-ADME .Calculations were carried out on a PC running at 3.4 GHz on an Intel i7 2600 quad core processor with 8 GB RAM and 1 TB hard disk with Windows 7 Home Premium 64-bit SP1 as an operating system. Ligands were built and minimized using MMX force field by PC Model version 9.3 . The minimized molecules were saved in pdb extension. The structures of RNA-dependent RNA polymerase from SARS-CoV-2 bound to the template-primer RNA and phosphate form of remdesivir (PDB ID: 7BV2) was determined by electron microscopy with a resolution of 2.5 Å . The str+ and Cl− ions at final concentration of 0.9% (m/V) [Calculations were carried out on a PC running at maximum clock of 4.9 GHz on an AMD-Ryzen9 5950×, 16 core processor (32 threads) with 32 GB RAM and 1 TB hard disk with Windows 10 at 64-bit as an operating system by using Yasara software   The stru9% (m/V) , a steep9% (m/V) ,30 The p9% (m/V) ,30 The i6 simulation resulted in 16,104 atoms for the protein, 790 atoms in nucleic acid, 54 atoms in ligand 6, 177 Na+ ions, 149 Cl− ions and 57,093 molecules of water for a total of 188,553 atoms in the soup. The system composition for GS-441524 simulation resulted in 16,104 atoms in the protein, 790 atoms in nucleic acid, 36 atoms in GS-441524 ligand, 177 Na+ ions, 150 Cl− ions and 57,504 molecules of water for a total of 189,771 atoms in the soup.The system composition for compound 5 was built using PC Model version 9.3  and minimized by MM using MMX force field. Conformational analysis was achieved by GMMX search module present in PC Model. Gaussian 03W revision E.01 program [DFT calculation was performed in the gas phase and in chloroform by using Polarized Continuum Model (PCM)  Calculat program  with gra program  for the  program ,35,36 wa6, emerging as the most promising structure in terms of pharmacokinetic parameters and by docking calculation, MD simulation showed a stable molecular interaction in RdRp complex and a minor average atomic fluctuation (RMSF) than remdesivir metabolite, suggesting a well accommodation in the RdRp pocket.A series of new enantiomerically pure nucleoside analogues were synthesized from the chiral building block LAC obtained as one added value product from cellulose pyrolysis in the context of biorefinery. By a convergent synthetic approach, seven molecules showing modifications on the nucleobase and on the carboxyl moiety on the tetrahydrofurane ring have been obtained through a Mitsunobu substitution where NOE experiments proved the configurational inversion supported by energy-minimized structure DFT calculation. ADME and drug-likeness prediction showed favorable parameters in comparison with the recently approved anti COVID-19 agent remdesivir and its metabolite GS-441524. Docking calculation took into account the complex system formed by RdRp enzyme and each small molecule covalently linked to primer RNA. For compound 6 as a promising candidate to deepen the study on a potential therapeutic inhibitor of SARS-CoV-2 RNA-dependent RNA polymerase. These findings emphasize the interest to deepen the study in next biological evaluation.In summary, the whole computational study provided data also better than remdesivir metabolite, all agree in indicating compound


Holothuria spinifera led to the isolation of a new cerebroside, holospiniferoside (1), together with thymidine (2), methyl-α-d-glucopyranoside (3), a new triacylglycerol (4), and cholesterol (5). Their chemical structures were established by NMR and mass spectrometric analysis, including gas chromatography–mass spectrometry (GC–MS) and high-resolution mass spectrometry (HRMS). All the isolated compounds are reported in this species for the first time. Moreover, compound 1 exhibited promising in vitro antiproliferative effect on the human breast cancer cell line (MCF-7) with IC50 of 20.6 µM compared to the IC50 of 15.3 µM for the drug cisplatin. To predict the possible mechanism underlying the cytotoxicity of compound 1, a docking study was performed to elucidate its binding interactions with the active site of the protein Mdm2–p53. Compound 1 displayed an apoptotic activity via strong interaction with the active site of the target protein. This study highlights the importance of marine natural products in the design of new anticancer agents.Chemical investigation of the methanolic extract of the Red Sea cucumber  Holothuria, is the most predominant genus reported in the World Register of Marine Species database +  of 1 indicated typical signals of an aliphatic chain, overlapped methyls at δH 0.85, and long methylene chain protons at δH 1.29, besides an amide group exchangeable protons at δH 8.58 , showing a sphingolipid backbone. Additionally, several characteristic resonances were noticed at δH 5.27 , 4.20 , 4.35 , 4.34, 4.54 , and 4.74  as well as a signal for an anomeric proton at δH 4.96 . The 13C NMR spectrum  displayed resonances for an amide carbonyl at δC 175.7 and two terminal methyl groups in aliphatic hydrocarbon chains at δC 14.1, and it exhibited characteristic signals at δC 51.6 (C-2), 70.4 (C-1), 72.3 (C-4), 72.4 (C-2’), and 75.7 (C-3) corresponding to a 2-amino-1,3,4,2’-tetrol part of the compound. Besides, an anomeric carbon at δC 105.5, in addition to other downfield-shifted resonances at δC 75.0, 78.3, 71.3, 78.4, and 62.5, was present, indicating a sugar part. The configuration of the glucopyranosyl moiety was determined to be β-configured according to the noticed coupling constant of the anomeric proton at δH 4.96 , indicating the diaxial coupling between H-1′’ and H-2′’ as well as the chemical shift of the anomeric carbon δC 105.5 ppm  ), it was subjected to gas chromatography–mass spectrometry (GC–MS) analysis after oxidative cleavage by KMnO4 as described in  fragment; m/z 253, corresponding to [C16H29O2•]; and m/z 125, ascribed to [C9H17•] (E)-2-hydroxytricos-15-enoate. To identify the sugar moiety in compound 1, the resulting sugar derivative from methanolysis was scanned by HPLC, and its retention time (tR) was compared with those of standard sugars. The obtained results revealed that the unknown sugar was glucose (tR = 4.62 min). Additionally, the acetylated thiazolidine derivative of the obtained glucose from methanolysis was inspected by reversed-phase HPLC, and it exhibited the exact tR of the acetylated thiazolidine analogue of standard D-glucose (19.7 min). The configuration of the cerebroside moiety was achieved via comparison of its 1H NMR, 13C NMR (measured in C5D5N), and physical data with those of the analogues reported previously, in which the chemical shifts of C-2 (δC 51.6), C-3 (δC 75.7), C-4 (δC 72.3), and C-2’ (δC 72.4), together with the chemical shifts of their corresponding protons besides the optical rotation +17.40 , were in congruence with those of the known reported cerebroside SJC-3 + , and 1H ed in −1  20] The The1 (di8.5 ppm) ,22. The n et al. . Brieflyter of 1  displaye[C9H17•] . Hence, 2  and (C–O–C), respectively +) and (Z)-9-octadecenoic acid methyl ester (oleic acid methyl ester) C19H36O2 (m/z 296 [M]+). The geometry of the double bonds was further confirmed by the chemical shifts of the allylic carbons at δC 27.1 and 27.2, which characterize the (Z)-configuration of the double bond + . Its molectively   was assessed on breast cancer cell line (MCF-7) by the sulforhodamine B (SRB) assay using the method described in +; 1H NMR  and 13C NMR  spectral data, see White powder; HRMS: 1H NMR : δH 8.14 , 7.03 , 4.98 , 4.46 , 4.24 , 4.15 , 2.66–2.60 , 1.85 ; 13C NMR : δC 164.9 (C-4), 151.9 (C-2), 136.6 (C-6), 110.4 (C-5), 88.8 (C-4’), 85.2 (C-1’), 71.4 (C-3’), 62.2 (C-5’), 41.3 (C-2’), 12.7 .White amorphous powder; 1H NMR : δH 5.15 , 4.24 , 4.13 , 4.21, 3.51, 4.36 , 4.50 , 3.44 ; δc 101.4 (C-1), 73.8 (C-2), 75.4 (C-3), 72.1 (C-4), 74.1 (C-5), 62.8 (C-6), 54.8 (OCH3).White amorphous powder; m/z 871.7609 [M + H]+. 1H NMR  and 13C NMR  spectral data, see White powder; HRMS: 1H NMR : δH 5.36 , 3.53 , 2.27 , 2.00 , 1.86 , 1.50 , 1.15–2.33 , 1.02 , 0.93 , 0.88 , 0.85 , 0.70 ; 13C NMR : δC 140.8 (C-5), 121.7 (C-6), 71.8 (C-3), 50.1 (C-9), 42.3 (C-4), 39.8 (C-12), 39.8 (C-24), 37.2 (C-1), 36.5 (C-10), 36.2 (C-20), 33.9 (C-22), 31.6 (C-2), 31.6 (C-7), 31.6 (C-8), 28.9 (C-25), 23.9 (C-23), 21.1 (C-11), 19.6 (C-27), 19.4 (C-26), 19.0 (C-19), 18.8 (C-21), 11.7 (C-18).White amorphous powder; 1 on MCF-7 breast cancer cell line was evaluated by the SRB assay according to the method previously mentioned in Abdelhameed et al. and Skehan et al. [50 values of the test compounds were calculated.The cytotoxicity of compound n et al. ,35 using1 coordinate was established applying ChemDraw Ultra 11.0. After that, its protonation, correction of atoms, and bond types were clearly settled; hydrogen atoms were placed and protonation and finally minimization was carried out . The docking investigation was confirmed by redocking the ligand in PDB structure 3LBK, and the ligand was then removed. The default Triangle Matcher placement procedure was chosen for molecular simulation. GBVI/WSA dG scoring function, which estimates the free energy of binding of the ligand from a given pose, was selected to order the final poses. The ligand complex with the protein possessing the lowest S-score was picked out. The redocking of the ligand with its target exhibited a RMSD value of 0.806 Å, which verifies that the ligand binds to the same pocket and confirms the dependability of docking parameters.In the Protein DataBank, the crystallographic structures of 3LBK in complex with its ligand is accessible . StructuH. spinifra resulted in the isolation of the new cerebroside holospiniferoside (1), a new triacylglycerol, and three known compounds 2, 3, and 5. All the isolated compounds are reported in the Red Sea cucumber Holothuria spinifera for the first time. Holospiniferoside (1) exhibited promising cytotoxic potential against MCF-7 cancer cells (IC50 = 20.6 µM). Moreover, it displayed strong interaction with the active site of the Mdm2–p53 protein, suggesting its apoptotic activity. In brief, marine organisms could be considered as gold mines of promising anticancer drugs.In continuation of our research toward the isolation of bioactive metabolites of marine origin, we herein report that the phytochemical investigation of the sea cucumber


Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4′ coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8′ coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In  The dirigent protein AtDP1/AtDIR12 and the laccase AtLAC5 play essential roles in the biosynthesis of seed-coat protective neolignans via sinapoylcholine/feruloylcholine in Arabidopsis. Phenolic compounds including lignins, lignans, neolignans, and flavonoids are major plant secondary metabolites 2] with a C8-C8′ linkage; compounds with all other types of linkages are called neolignans , pinoresinol is converted to lariciresinol by pinoresinol reductase -pinoresinol, a lignan, in thia sp. . Subsequthia sp. . DIR-medium spp. , 2017. Mium spp. .DIRs belong to a multi-gene family that is classified into six subfamilies  based on phylogenetic analyses . The DIRAtDIR1–AtDIR25) that are distributed in the DIR-a, DIR-b/d, and DIR-e subfamilies in addition to an atypical type, OVULE ABORTION 2  in the DIR-e subfamily is essential for building the extracellular lignin-based Casparian strip , that showed no significant correlation with putative lignans and neolignans that accumulate in roots and other DIR genes -1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine, SC(4-O-8)G  cells of seeds. In addition, we found that mutants deficient in AtLAC5, a LAC co-expressed with AtDP1/AtDIR12 showed significant metabolic changes among feruloylcholine (FC)-conjugated neolignans, including erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3-methoxyphenyl}acryloylcholine, FC(4-O-8)G -conjugated neolignan, -8)G 1e;  as the c)G 4e in . Neoligndp1 (SAIL_60_D04) lacked a compound having a peak at m/z 668 in seeds  was renamed atdp1-1 (AtDP1/AtDIR12 promoter region in atdp1-2. Reverse transcription-polymerase chain reaction (RT-PCR) analysis detected AtDP1/AtDIR12 transcripts in atdp1-2, whereas the transcript was not detected in atdp1-1 . Independent transgenic lines carrying gAtDP1-1 and gAtDP1-2 had essentially the same metabolic profile as wild-type plants, although the levels of the target compound were lower in transgenic lines carrying gAtDP1-2  missing in the atdp1-1 mutant was proposed to be 3-{4-[2-hydroxy-2-(4-hexosyloxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine, SC(4-O-8)G 4-O-Hex  was proposed to be 3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine, SC(4-O-8)G G 1; .atdp1 mutant, we chemically synthesized authentic standards (racemates) for the two stereoisomers of neolignan 1, namely erythro-SC(4-O-8)G (1e) and threo-SC(4-O-8)G (1t), as shown in 1H and 13C and 2D correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond coherence (HMBC) pulse schemes  in the atdp1-1 mutant were identical to those of the authentic standard 1e (O-8)G (1) previously isolated from transgenic Brassica napus seeds G (4), FC(5-8)G (6), and FC(5-8)G 4-O-Hex G (4e) and threo-FC(4-O-8)G (4t), respectively, in a manner similar to that described above for the SC(4-O-8)G isomers 1e and 1t (O-8)G depleted in the atdp1-1 mutant was the erythro isomer 4e (O-8)G, the Col-0 seeds exclusively contain the erythro isomer 4e with no detectable threo isomer 4t  was also detected. Taken together, our results indicate that AtDP1/AtDIR12 specifically mediates the formation of SC- and FC-conjugated 8-O-4′-type neolignans 1e and 4e in Arabidopsis. In addition, the depletion of FC(5-8)G (6) and FC(5-8)G 4-O-Hex (7) in the atdp1-1 mutant suggested that AtDP1/AtDIR12 may also be involved in the formation of FC-conjugated 8-5′-type neolignans. Interestingly, the level of SC(4-O-8)S (3) is higher in the atdp1-1 mutant  involved in SC(4-O-8)S biosynthesis.Further comprehensive analyses of neolignans, and SC and FC derivatives suggested that, along with the SC-conjugated neolignan O-Hex 7; ] in the ild type . Accordisomer 4e . As we osomer 4t . A putat1 mutant . This fiForsythia intermedia, western red cedar (Thuja plicata), Arabidopsis, Schizandra chinensis, pea (Pisum sativa), and flax (Linum usitatissimum) were previously shown to be involved in the biosynthesis of lignans, (+)- or (−)-pinoresinol, via 8-8′ phenoxy radical coupling and belong to the DIR-a subfamily - and/or (−)-pinoresinol-forming DIRs  were found . Of the AtDP1/AtDIR12 transcripts in various organs including seeds at nine developmental stages was investigated by reverse transcription quantitative PCR (RT-qPCR). AtDP1/AtDIR12 transcripts predominantly accumulated in seeds at developmental stages 9 to 11, but not in other organs (erythro-SC(4-O-8)G (1e) and the expression profile of AtDP1/AtDIR12 archived on the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). The expression profile of anthocyanidin reductase encoded by BANYULS (BAN), a key enzyme in the proanthocyanidin (PA) biosynthetic pathway, was clearly distinct from that of AtDP1/AtDIR12 during seed development. Compared to AtDP1/AtDIR12, BAN transcripts accumulated in seeds across a broader range of developmental stages  were generated. The YFP fluorescence was specifically observed in the outer integument 1 (oi1) of seeds expressing ProAtDP1-AtDP1CDS-YFP (http://www.cbs.dtu.dk/services/TargetP/) predicts the presence of an N-terminal signal peptide in AtDP1/AtDIR12 (likelihood 0.9957), suggesting that AtDP1/AtDIR12 may sort to compartments in the secretory pathway.To assess the cellular localization patterns of AtDP1/AtDIR12 in developing seeds, transgenic plants harboring a 720-bp promoter region of 1CDS-YFP . Additio1CDS-YFP . These d1CDS-YFP . TargetPForsythia DIR  of coordinated expression were noted between AtDP1/AtDIR12 and two LACs, i.e. AtLAC15/TRANSPARENT TESTA 10  and AtLAC5 . AtLAC15/TT10 and AtLAC5 are major LACs expressed in seeds -pinoresinol in the presence of thia DIR . Therefoin seeds . RT-qPCRC15/TT10 .LACs have been functionally identified as LACs that polymerize lignin , and SALK_128292 , and SALK_093534 (lac5-3), were isolated. No transcripts of AtLAC5 in the lac5-2 and lac5-3 lines were found by RT-PCR analysis G (4e), FC(5-8)G (6), and FC(5-8)G 4-O-Hex G (1e) in the lac5 mutants may be slightly lower than that in wild type, but the accumulation level was much higher than that in the atdp1 mutant. Neolignans in tt10 mutants showed no significant changes in abundance. The content of choline derivatives -GUS fusion construct suggested that AtLAC5 is localized in the replum and abscission zone in siliques and seed embryos . The blue coloration by GUS staining was predominantly observed in the replum, septum, and funiculus and to a lesser extent in the valves of transgenic plants, using either promoter  predicts the presence of an N-terminal signal peptide in AtLAC5 (likelihood 0.9998), suggesting that AtLAC5 may sort to compartments in the secretory pathway as is also the case for AtDP1/AtDIR12. Transcriptome data obtained from the Arabidopsis eFP browser showed that AtLAC5 transcripts accumulate exclusively in seeds and particularly in seed coats of mature green seeds . SC levels are decreased and levels of 1-O-sinapoyl-β-d-glucose and choline are increased in ethyl methanesulfonate-induced sng2 mutant seeds  and sng2-4 (SALK_018120C) to compare with a known sng2-2 mutant  may directly couple with CA via 8-ouplings . SC is s choline . SCT/SCP choline . A mutannt seeds . We isolC and FC . On the 2 mutant . Collectolignans .transparent testa mutants that are unable to synthesize flavonoids, including PAs, showed a significant increase in seed coat permeability and reduced germination capacity during long-term seed storage , also had reduced yields of guaiacyl-type lignin monomers, whereas yields of syringyl-type lignin monomers in the lac5 mutants were similar to those of wild type  and/or  configurations, are yet to be determined, it is plausible that AtDP1/AtDIR12 guides the regio- and stereo-selective 8-O-4′ coupling of CA with SC and FC to form optically active products. Intriguingly, the wild-type seeds appeared to accumulate the erythro isomers 1e and 4e exclusively with no detectable amounts of the corresponding threo isomers 1t and 4t - and (−)-pinoresinol-forming DIRs. AtDP1/AtDIR12, AtDIR13, and AtDIR14 belong to a cluster of (−)-pinoresinol-forming DIRs that include AtDIR6 and AtDIR5  8-8′ coupling of CA to produce lignan, (+) or (−)-pinoresinol, have been identified , but thed AtDIR5 . Evolutid AtDIR5 . These dsng2 mutants that are defective in the ability to synthesize SC revealed that SC- and FC-conjugated neolignans are synthesized from SC/FC and CA, and AtDP1/AtDIR12 is essential for producing the 8-O-4′-type neolignans, erythro-SC(4-O-8)G (1e) and erythro-FC(4-O-8)G (4e), in Arabidopsis seeds. Notably, in seeds of the atdp1-1 mutant, the abundance of other neolignans putatively annotated as FC-monolignol conjugate FC(5-8)G (6) and its glycoside FC(5-8)G 4-O-Hex G from ferulic acid and CA via ferulic acid(4-O-8)G and feruloylhexose(4-O-8)G has been suggested , lignans are mainly localized in the secondary walls of sclerite cells in the seed outer integument  are localized to the cell wall lignans, including aglycones and glycosides, are reportedly stored in Arabidopsis leaf vacuoles . On the tegument . AtDP1/Ategument . In Forsell wall . Arabidoell wall , and AtLell wall . Serine ell wall . FurtherO-4′-type as well as the 8-5′-type neolignans but is not involved in the synthesis of SC-conjugated neolignans  mutants. Our results indicated that TT10/AtLAC15 is not involved in neolignan biosynthesis and suggest that the substrate specificity of TT10/AtLAC15 may be also strict in vivo, or TT10/AtLAC15 may be separated spatially from monolignols in the cells.The pe seeds . TT10 ispe seeds ; howeverAtDP1/AtDIR12 is specifically expressed in seeds. Analyses using transgenic plants harboring an AtDP1/AtDIR12 promoter fused to AtDP1/AtDIR12 cDNA with YFP or GUS showed that AtDP1/AtDIR12 is localized in the oi1 cells of developing seeds  was used as the wild type in this study. The T-DNA inserted mutants, SAIL_60_D04 (atdp1-1) and SALK_062238 (atdp1-2) for AtDP1/AtDIR12, SALK_002972 (tt10-4) and SALK_128292 (tt10-5) for AtLAC15/TT10, SALK_092440 (lac5-2) and SALK_093534 (lac5-3) for AtLAC5, SALK_002255C (sng2-2), SALK_053495C (sng2-3), and SALK_018120C (sng2-4) for SNG2 were obtained from the Arabidopsis Biological Resource Center . As the parental line of SAIL_60_D04 (atdp1-1), CS8846 was used for comparison. T-DNA insertion lines, SALK_062238 (atdp1-2), SALK_092440 (lac5-2), SALK_093534 (lac5-3), SALK_053495C (sng2-3), and SALK_018120C (sng2-4) were screened by PCR using specific primers for AtDP1/AtDIR12 (At4g11180-6818953f and At4g11180-6819803r), AtLAC5 (At2g40370/886f and At2g40370/1826r), SNG2 (At5g09640_SCPL19_-8F and At5g09640_SCPL19_2519R), and T-DNA , SALK_002972 (tt10-4), SALK_128292 (tt10-5), and SALK_002255C (sng2-2), were isolated as described previously (−2 s−1 of fluorescent lamp)/dark cycle. mutants . Homozygeviously . Plants AtDP1/AtDIR12 coding region and 0.6 kb of the 3′-noncoding region were amplified by PCR using primers, gDP1-Rv2 and CACC-gDP1-Fw1 or CACC-gDP1-Fw2 . gDP1-1  and gDP1-2  were transformed into Agrobacterium tumefaciens strain EHA105, and Arabidopsis plants were transformed by the floral-dip method , 50 mg L−1 kanamycin sulfate, and 50 mg L−1 carbenicillin, sodium salt.For complementation tests, ca. 3.0-kb and 1.9-kb genomic fragments covering 1.8 kb and 0.72 kb of the promoter region, the entire gDP1-Fw2 . Amplifip method . T2 plan−1 lidocaine, and 10-camphor sulfonic acid, using a mixer mill  with zirconia beads for 10 min at 20 Hz. After centrifugation at 15,000g and filtration , the extracts (3 µL) were applied to a liquid chromatography (LC)–mass spectrometry (MS) system with an electrospray ionization (ESI) interface . For analyses of the putative neolignan compounds (m/z = 668 or 506), LC system conditions  were as described previously (The seeds (3.0 mg) were homogenized in 50 volumes of the extraction solvent, 50% (v/v) MeOH with 0.5-mg Leviously .m/z 50‒1,500; scan duration, 0.5 s; interscan delay, 0.014 s; data acquisition, centroid mode; polarity, positive; Lockspray : scan duration, 1.0 s; interscan delay, 0.1 s. MS/MS data were acquired in the ramp mode with the following analytical conditions: (1) MS: mass range, m/z 50–1,500; scan duration, 0.5 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; polarity, positive and (2) MS/MS: mass range, m/z 50–1,500; scan duration, 0.1 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; polarity, positive; collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra of the top 10 ions  in an MS scan were automatically obtained. If the ion intensity was less than 1,000, MS/MS data were not collected and the instrument moved to the next top 10 ions. Original peak intensity values were divided with those of the internal standard  to normalize the peak intensity values and were shown as relative peak intensities.For analyses of neolignans and choline derivatives, the dried samples were extracted with 50 µL of 80% (v/v) MeOH containing 2.5 mM lidocaine and 10-camphor sulfonic acid per mg dry weight using a mixer mill with zirconia beads for 7 min at 18 Hz and 4°C. After centrifugation for 10 min, the supernatant fraction was filtered using an HLB µElution plate (Waters). The extracts (1 µL) were analyzed using an LC–Q-Tof–MS . Analytical conditions were as follows: LC column, Acquity bridged ethyl hybrid (BEH) C18 ; solvent system, solvent A [water including 0.1% (v/v) formic acid] and solvent B [acetonitrile including 0.1% (v/v) formic acid]; gradient program, 99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 75%A/25%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 99.5%A/0.5%B at 12.1 min, and 99.5%A/0.5%B at 15.0 min; flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min, and 0.3 mL/min at 14.5 min; column temperature, 40°C; MS detection: capillary voltage, +3.0 keV, cone voltage, 25.0 V, source temperature, 120°C, desolvation temperature, 450°C, cone gas flow, 50 L/h; desolvation gas flow, 800 L/h; collision energy, 6 V; mass range, O-8)G (1) and FC(4-O-8)G (4) were synthesized as shown in 8SC and ethyl ferulate 8FC were synthesized according to methods described in the literature . Standard 1D and 2D  NMR experiments were used for structural assignments. Determination of erythro and threo configurations was based on the comparison of NMR chemical shifts and coupling constant data reported for identical or analogous erythro and threo 8-O-4-type dimer models as reported by The authentic, racemic standards for neolignans SC, dried over Na2SO4, and evaporated in vacuo. The crude product was then purified by flash chromatography to produce a white solid of compound 9 (48.9% yield). 1H-NMR : δ = 7.74 , 7.73 , 7.27 , 4.82 , 3.95 , 2.31 .To a stirred solution of 1-(4-acetoxy-3-methoxyphenyl)-1-ethanone (9.6 mmol) in EtOAc (10 mL), pyridinium bromide perbromide (9.6 mmol) was added. After 2 h of stirring at room temperature, the reaction mixture was neutralized with 1 M NaHCO8SC (3.48 mmol) and K2CO3 (3.48 mmol) in 10 mL acetone, compound 9 (3.87 mmol) in 20 mL acetone was added dropwise at room temperature and then refluxed for 16 h. After cooling to room temperature, the reaction mixture was filtered and added to EtOAc/hexane . The organic phase was washed with distilled water and brine, dried over Na2SO4, and evaporated in vacuo to a residue that was purified by flash chromatography to produce a colorless solid of compound 10SC (57.4% yield). 1H-NMR : δ = 7.70 , 7.63  7.60 , 7.13 , 6.75 , 6.35 , 5.24 , 4.27 , 3.90 , 3.84 , 2.33 , 1.34 .To a suspension of compound 10FC was synthesized from compounds 8FC and 9 using the above described procedure for compound 10SC and was isolated as a colorless solid (73.3% yield). 1H-NMR : δ = 7.66 , 7.64-7.56 , 7.16 , 7.08 , 7.04 , 6.78 , 6.32 , 5.37 , 4.26 , 3.93 , 3.90 , 2.35 , 1.34 .Compound 10SC (1.96 mmol) in 30 mL of dioxane, K2CO3 (14 mmol) and 37% (v/v) formaldehyde (3.92 mmol) were added. After 5 h of stirring at room temperature, the reaction mixture was filtered and concentrated in vacuo to a residue that was purified by flash chromatography to produce a colorless solid of compound 11SC (76.2% yield). 1H-NMR : δ = 7.72 , 7.67 , 7.58 , 7.12 , 6.75 , 6.35 , 5.15–5.16 , 4.26 , 3.98–4.04 , 3.89 , 3.76 , 2.34 , 1.33 .To a stirred suspension of compound 11FC was synthesized from compound 10FC using the above described procedure for compound 11SC and was isolated as colorless solid (86.3% yield). 1H-NMR : δ = 7.70 , 7.68 , 7.58 , 7.14 , 7.06 , 7.00 , 6.75 , 6.30 , 5.52 , 4.25 , 4.08–4.16 , 3.87 , 3.86 , 2.34 , 1.33 .Compound 12SC (1.45 mmol) in 25 mL ethanol, NaBH4 (10.5 mmol) was added at room temperature. The reaction mixture was stirred under nitrogen gas at room temperature for 5 min, and then the excessive NaBH4 was quenched by adding acetic acid (1 mL). The reaction mixture was concentrated in vacuo and extracted three times with EtOAc (10 mL) over water (10 mL). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated in vacuo to a crude oil that was purified by flash chromatography to produce a colorless solid of compound 12SC as a mixture of erythro and threo isomers . 12SC (erythro isomer): 1H-NMR : δ  =  7.65 , 7.25 , 7.13 , 7.08–7.11 , 7.01–7.04 , 6.53 , 5.12 , 4.29–4.34 , 4.25 , 3.96 , 3.87 , 3.76–3.82 , 3.49–3.58 , 2.27 , 1.33 . 12SC (threo isomer): 1H-NMR δH (acetone-d6) δ  =  7.65 , 7.27 , 7.13 , 7.08–7.11 , 7.01–7.04 , 6.58 , 5.16 , 4.25 , 4.16–4.20 , 3.99 , 3.84 , 3.61–3.70 , 3.40–3.48 , 2.27 , 1.33 .To a solution of compound 12FC was synthesized from 11FC using the above described procedure for compound 12SC and was isolated as a mixture of erythro and threo isomers in the form of a colorless solid . 12FC (erythro isomer): 1H-NMR : δ  =  7.65 , 7.37 , 7.33 , 7.07–7.25 , 6.47 , 5.07 , 4.49–4.53 , 4.21–4.29 , 3.98 , 3.94 , 3.82–3.90 , 3.61–3.65 , 2.27 , 1.33 . 12FC (threo isomer): 1H-NMR : δ  =  7.63 , 7.41 , 7.33 , 7.07–7.25 , 6.49 , 5.07 , 4.54–4.58 , 4.21–4.29 , 3.98 , 3.94 , 3.82–3.90 , 3.61–3.65 , 2.27 , 1.33 .Compound 12SC (1.12 mmol) was dissolved in 20 mL of 2 M NaOH. After stirring for 10 min at room temperature, the reaction mixture was acidified with 3 M HCl, and extracted three times with CH2Cl2-EtOAc . The combined organic layer was washed with brine, dried over Na2SO4, and evaporated in vacuo. The crude product was purified by flash chromatography to give a colorless oil of compound 13SC as a mixture of erythro and threo isomers . 13SC (erythro isomer): 1H-NMR : δ  =  7.63 , 7.02 , 6.96 , 6.83 , 6.78 , 6.47 , 5.04 , 4.36–4.39 , 3.95–3.98 , 3.90 , 3.86 , 3.63–3.67 . 13SC (threo isomer): 1H-NMR : δ  =  7.64 , 7.06 , 7.02 , 6.91 , 6.79 , 6.49 , 5.04 , 4.18–4.22 , 3.82–3.85 , 3.93 , 3.87 , 3.40–3.42 .Compound 13FC was synthesized from 12FC using the above described procedure for compound 7SC and was isolated as a mixture of erythro and threo isomers in the form of a colorless oil . 13FC (erythro isomer): 1H-NMR : δ  =  7.62 , 7.36 , 7.21 , 7.12–7.14 , 6.92 , 6.78 , 6.44 , 4.91–4.92 , 4.37–4.41 , 3.95 , 3.83 , 3.72–3.77 , 3.53–3.58 . 13FC (threo isomer): 1H-NMR : δ  =  7.58 , 7.31 , 7.19 , 7.12–7.14 , 7.05 , 6.92 , 6.76 , 6.42 , 4.91–4.92 , 4.46–4.49 , 3.95 , 3.83 , 3.72–3.77 , 3.53–3.58 .Compound 13SC was carried out according to the method of 13SC (0.116 mmol) in DMF (220 µL), 1 M NaOH (110 µL), and 1 M bromocholine bromide in water (220 µL) were successively added at room temperature. The reaction mixture was then heated to 90°C for 24 h. After cooling to room temperature, the reaction mixture was diluted with water (2 mL), eluted on a Strata-X-CW solid-phase extraction cartridge , and concentrated in vacuo, to give a crude mixture of erythro and threo isomers 1e and 1t as a colorless oil (yield 65.6%). A portion of this crude mixture was dissolved in H2O-MeOH , and subjected to preparative HPLC to isolate pure compounds 1e and 1t. erythro-SC(4-O-8)G (1e): 1H-NMR : δ  =  7.70 , 6.99 , 6.95 , 6.79 , 6.73 , 6.55 , 4.90 , 4.69-4.65 , 4.37–4.34 , 3.91 , 3.85 , 3.83 , 3.79–3.77 , 3.62 , 3.26 ; 13C-NMR : δ  =  167.47 (C-9), 154.80 (C-3′/5′), 148.68 (C-3), 147.53 (C-7′), 146.96 (C-4), 139.57 (C-4′), 133.85 (C-1), 131.27 (C-1′), 120.80 (C-6), 117.11 (C-8′), 115.68 (C-5), 111.58 (C-2), 107.01 (C-2′/6′), 87. 52 (C-8), 74.18 (C-7), 66.25 (C-11′), 61.80 (C-9), 58.89 (C-10′), 56.80 (C-3′/5′OMe), 56.37 (C-3OMe), 54.48 (C-12′NMe). threo-SC(4-O-8)G (1t): 1H-NMR : δ  =  7.71 , 7.01 , 6.98 , 6.87 , 6.75 , 6.57 , 4.99 , 4.69–4.65 , 4.22–4.19 , 3.99 , 3.83 , 3.80–3.76 , 3.37 , 3.26 ; 13C-NMR : δ  =  167.49 (C-9), 154.60 (C-3′/5′), 148.75 (C-3), 147.21 (C-7′), 147.21 (C-4), 139.96 (C-4′), 133.54 (C-1), 131.43 (C-1′), 120.83 (C-6), 117.30 (C-8′), 115.84 (C-5), 111.77 (C-2), 106.99 (C-2′/6′), 88. 88 (C-8), 74.41 (C-7), 66.24 (C-11′), 61.92 (C-9), 58.92 (C-10′), 56.83 (C-3′/5′OMe), 56.38 (C-3OMe), 54.51 (C-12′NMe).Choline esterification of compound 4 as a crude mixture of erythro and threo isomers 4e and 4t  was prepared from 13FC using the above described procedure for compound 1. Pure isomers 4e and 4t were then isolated by preparative HPLC. erythro-FC(4-O-8)G (4e): 1H-NMR : δ  =  7.66 , 7.18 , 7.10 , 7.03 , 6.97 , 6.84 , 6.70 , 6.45 , 4.81 , 4.67–4.64 , 4.51 , 3.84 , 3.83 , 3.82 , 3.80 , 3.79–3.76 , 3.25 ; 13C-NMR : δ  =  167.70 (C9′), 152.24 (C-4′), 151.82 (C-3′), 148.67 (C-3), 147.58 (C-7′), 147.10 (C-4), 133.99 (C-1), 129.14 (C-1′), 123.81 (C-6′), 121.17 (C-6), 117.40 (C-5′), 115.61 (C-5), 115.46 (C-8′), 112.54 (C-2′), 112.06 (C-2), 85.47 (C-8), 74.11 (C-7), 66.27 (C-11′), 62.48 (C-9), 58.78 (C-10′), 56.63 (C-3′OMe), 56.34 (C-3OMe), 54.47 (C-12′NMe). threo-FC(4-O-8)G (4t): 1H-NMR : δ  =  7.69 , 7.25 , 7.14 , 7.06 , 7.03 , 6.85 , 6.75 , 6.47 , 4.89 , 4.68–4.63 , 4.48–4.45 , 3.90 , 3.82 , 3.79–3.75 , 3.53 , 3.25 ; 13C-NMR : δ  =  167.69 (C9′), 152.49 (C-4′), 151.74 (C-3′), 148.85 (C-3), 147.53 (C-7′), 147.22 (C-4), 133.81 (C-1), 129.27 (C-1′), 123.96 (C-6′), 120.66 (C-6), 117.40 (C-5′), 115.85 (C-5), 115.60 (C-8′), 112.51 (C-2′), 111.74 (C-2), 86. 21 (C-8), 73.91 (C-7), 66.27 (C-11′), 62.09 (C-9), 58.80 (C-10′), 56.68 (C-3′OMe), 56.35 (C-3OMe), 54.48 (C-12′NMe).Compound https://www.megasoftware.net/; Deduced amino acid sequences of DIRs or LACs were aligned using the CLUSTAL W program in MEGA X  and were checked for specific product formation using a dissociation program. Plasmid DNAs containing the corresponding genes were used as templates for calibration. RT-qPCR was performed in triplicate.RNA extraction, cDNA synthesis, and reverse transcription quantitative PCR (RT-qPCR) were performed as described previously . The devAtDP1/AtDIR12 promoter region or the promoter region including the AtDP1/AtDIR12 coding region was amplified by PCR using primers, At4g11180_CCAC+promoter F and At4g11180_promoter R or At4g11180_R (TMII enzyme mix (Invitrogen).A 720-bp fragment of the g11180_R . Amplifig11180_R  and pBGGAtLAC5 promoter region were amplified by PCR using primers, At2g40370pro_CCAC + 4140F or At2g40370pro_CCAC + 2000F and At2g40370ATG_R ; Carl Zeiss) with 488-nm excitation and a 505–530 nm band-pass filter for YFP, 488-nm excitation and a 650-nm long-pass filter for chlorophyll autofluorescence and a 25-mV argon laser. Composite figures were prepared using the Zeiss LSM Image Browser software.−1 5-bromo-4-chloro-3-indolyl glucuronide, 2 mM potassium ferricyanide, and 2 mM potassium ferrocyanide. After 60 min vacuum infiltration, samples were incubated overnight at 37°C and subsequently de-stained by a series of washes in 70% (v/v) ethanol, ethanol/acetic acid (6:1 v/v ratio), 70% (v/v) ethanol. For sectioning, tissue samples were fixed with 2% (v/v) glutaraldehyde and 1% (v/v) formaldehyde in 0.1 M phosphate buffer, pH 7.0, for 24 h at 4°C, washed in phosphate buffer, dehydrated using a series of graded ethanol solutions  and embedded in Technovit 7100 resin  according to the manufacturer’s instructions. Tissue sections were sliced into 5-µm transverse sections using a rotary microtome (Leica RM2125) and tungsten carbide disposable blades (Leica TC-65). Sections were observed using an Olympus BX53 microscope.For histochemical GUS assays, tissues were treated with ice-cold 90% (v/v) acetone for 20 min on ice and stained in 100 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100, 0.5 mg mLSurfaces of dry seeds were observed using a scanning electron microscope (Hitachi TM-1000).r > 0.3) were extracted from the list of genes co-expressed with AtDP1/AtDIR12.Coexpression analyses were conducted as described previously Yonekura-. The geng for 3 min, the supernatant fraction was recovered and the absorbance at 485 nm was measured. Three biological replicates were used for the analyses.Seed coat permeability was tested by a tetrazolium penetration assay , 2014. D2S2O5 in a mixer mill (Qiagen Retsch MM300 TissueLyser) for 1 min at 20 Hz, followed by sonication for 20 min. After centrifugation at 15,000g for 5 min, the supernatant fraction was evaporated and resuspended in 1 mL of HCl: butanol: 70% acetone (2:10:3). The absorbance of the solutions before/after hydrolysis at 95°C for 60 min was measured at 545 nm and the difference was treated as the soluble PA fraction. The pellet after extraction with 70% acetone was also evaporated, suspended in the HCl: butanol: 70% acetone solution and hydrolyzed as the insoluble PA fraction.PA extraction and acid hydrolysis were performed in triplicate as described previously . Mature N,O-bis(trimethylsilyl)acetamide and quantified by gas chromatography/mass spectrometry using 4,4′-ethylenebisphenol as an internal standard (Mature seeds (40 mg) were pulverized, extracted successively with chloroform–methanol , 100% methanol, and water, followed by freeze-drying to obtain the seed cell-wall fractions . The seestandard .Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL libraries under the following accession numbers: AtDP1/AtDIR12 , AtDIR5 , AtDIR6 , AtDIR13 , AtDIR14 , FiDIR1 (AF210061), LuDIR1 (KM433751), LuDIR5 (KM433753), LuDIR6 (KM433752), PsDRR206 (U11716), ScDIR (HQ428029), TpDIR5 (AF210067), TpDIR6 (AF210070), AtLAC1 ; AtLAC2 ; AtLAC3 ; AtLAC4 ; AtLAC5 ; AtLAC6 ; AtLAC7 ; AtLAC8 ; AtLAC9 ; AtLAC10 ; AtLAC11 ; AtLAC12 ; AtLAC13 ; AtLAC14 ; AtLAC15 ; AtLAC16 ; AtLAC17 ; BdLAC5 ; BnTT10 (NP_001302959); GaLAC1 (NP_001316972); PtLAC3 (CAC14719); SofLAC (SCVPRZ3027A08.g and SCUTST3084C11.g); ZmLAC3 (CAJ30499).The following materials are available in the online version of this article.. Identification of neolignan FC(4-O-8)G (4) in Arabidopsis seeds. Multiple alignment of DIRs in the DIR-a family.. Multiple alignment of Arabidopsis LACs and functionally identified LACs.. T-DNA insertion mutants of AtLAC5.. Expression pattern of AtLAC5 and AtDP1/AtDIR12 using the Arabidopsis eFP browser ( browser  and Arab browser .. A proposed model for gene duplication of the Arabidopsis DIR-a gene family. Metabolites detected in seeds of wild-type and the tested mutants. Primers used in this study.  1H and 13C NMR spectra of synthetic neolignans SC(4-O-8)G and FC(4-O-8)G. One-way analysis of variance for  One-way analysis of variance for  One-way analysis of variance for  Alignment corresponding to the phylogenetic analysis in  Alignment corresponding to the phylogenetic analysis in koaa014_Supplementary_DataClick here for additional data file.


R,R)-8-8-17-18(, displayed the same activity in the Gram-negative strain used. Studies were performed on the inhibition of biofilm formation and quorum-sensing, to which the enantiomeric pair 8 displayed activity for the latter. To gain a better understanding of how the active compounds bind to the efflux pumps, docking studies were performed. Hit compounds were proposed for each activity, and it was shown that enantioselectivity was noticeable and must be considered, as enantiomers displayed differences in activity.Antimicrobial peptides are one of the lines of defense produced by several hosts in response to bacterial infections. Inspired by them and recent discoveries of xanthones as bacterial efflux pump inhibitors, chiral amides with a xanthone scaffold were planned to be potential antimicrobial adjuvants. The chiral derivatives of xanthones were obtained by peptide coupling reactions between suitable xanthones with enantiomerically pure building blocks, yielding derivatives with high enantiomeric purity. Among 18 compounds investigated for their antimicrobial activity against reference strains of bacteria and fungi, antibacterial activity for the tested strains was not found. Selected compounds were also evaluated for their potential to inhibit bacterial efflux pumps. Compound  Antimicrobial peptides are proteins produced by several hosts as a defense mechanism against bacteria. They are a response from the innate immune system, and to this day, still present effectiveness against a multiplicity of pathogenic microbes ,2. They Since these compounds possess an exposed cationic part and a hydrophobic core, they can establish an initial electrostatic interaction, allowing them to adsorb and aggregate in the negatively charged part of the membrane and also to insert into the membrane due to their hydrophobic moiety ,4. TheseAntimicrobial peptides have also been shown to display effective activity against other resistance mechanisms, such as biofilm ,14 and qDespite their usefulness, and since these peptides are part of a ubiquitous defense system for eukaryotic organisms, resistance to these compounds has already arisen ,18, maniAs such, strategies are being developed to obtain peptide-like molecules with lower manufacturing costs, higher stability, and able to retain activity in the presence of salts. The development of small molecule peptidomimetics has become an attractive field in this sense, and one example is the synthesis of amino acid substituted xanthones. In fact, the substitution of the hydrophobic xanthone core with cationic peptide moieties has shown promising results for the development of new antibacterial agents ,29. Our Regarding antimicrobial activity, it is important to highlight that the configuration of the stereogenic centers is often ignored, and only a few examples describe the activity for both enantiomers ,39. NeveHerein, new molecular modifications to the xanthone scaffold were planned in order to synthesize chiral peptide-like derivatives and investigate their antimicrobial activity, as well as mechanisms of antibacterial resistance, specifically inhibition of efflux pumps, biofilm formation, and quorum-sensing. The synergy of these compounds with a β-lactam antibiotic in a resistant strain of a clinically relevant bacterial species was also studied. Docking studies were performed to gain a better understanding of how these compounds could interact with relevant bacterial efflux pumps. For each chiral peptide-like derivative, both enantiomers were synthetized, in high enantiomeric purity, to explore enantioselectivity in all assays. The results obtained show the potential of these compounds acting in different mechanisms, suggesting these chiral peptide-like xanthones could be useful in tackling antimicrobial resistance.A library of 18 xanthone derivatives with different substitution patterns in one ring  or both rings  of the benzopyrano scaffold was synthesized, starting from hydroxy- or carbo1 has also been previously described, through a benzophenone intermediate, which produced the main product 3-methoxy-4-hydroxy-9H-xanthen-9-one (2), but also the secondary products 3,4-dihydroxy-9H-xanthen-9-one (3) and 3-hydroxy-4-methoxy-9H-xanthen-9-one (4), which were used for the synthesis of derivatives acetate (9) was prepared from 3-methoxy-4-hydro-9H-xanthen-9-one (2), using the same method as previously described for 2′-[bis(oxy)]diacetate (6), adjusting the amount of K2CO3 and BrCH2COOCH3 according to the number of hydroxyl groups of the chemical substrate and the stoichiometry of the reaction diacetic acid (7), adjusting the volume of the solution of 5M NaOH according to the number of acetate groups of the chemical substrate and the stoichiometry of the reaction [H-xanthen-4-yl)oxy]acetic acid (10) as white solid.Compound reaction , yieldinmax (cm−1) (KBr): 3204.0, 2925.0, 1758.6, 1604.1, 1585.2, 1467.8, 1290.4, 1229.6, 1033.1, 819.1, 766.6. 1H NMR  δ: 12.9 , 8.17 , 7.90 , 7.80 , 7.64 , 7.48 , 7.27 , 4.76 , 3.97 . 13C NMR  δ: 175.3 (C-9), 170.1 (COOH), 156.7 (C-3), 155.5 (C-4), 149.4 (C-1), 135.3 (C-10a), 134.3 (C-4a), 125.9 (C-2), 124.4 (C-6), 121.5 (C-8), 120.8 (C-7), 118.2 (C-5), 115.9 (C-8a), 109.8 (C-9a), 69.2 (OCH2), 56.6 (OCH3)  . HRMS (E01.07191 .S)-2-[(3-methoxy-9-oxo-9H-xanthen-4-yl)oxy]-N-(1-phenylethyl)acetamide [S)-11(] and (R)-2-[(3-methoxy-9-oxo-9H-xanthen-4-yl)oxy]-N-(1-phenylethyl)acetamide -N-(1-phenylethyl)acetamide [S)-11(]: Yield: 45%. m.p.: 164–167 °C. [α]D25 °C + 48.2 (c = 0.83 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 1.40) ((k: 1.40) . R)-2-[(3-Methoxy-9-oxo-9H-xanthen-4-yl)oxy]-N-(1-phenylethyl)acetamide [R)-11(]: Yield: 50%. m.p.: 165–167 °C. [α]D25 °C − 48.4 (c = 0.83 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 0.78) ((k: 0.78) . max (cm−1) (KBr): 3348.1, 1665.1, 1604.8, 1587.5, 1466.9, 1289.1, 1230.2, 1023.4, 817.7, 769.1. 1H NMR  δ: 8.38 , 8.17 , 7.96 , 7.82 , 7.50 , 7.46 , 7.45 – 7.27 , 7.26 , 5.10 , 4.65 , 3.93 , 1.48 . 13C NMR  δ: 175.2 (C-9), 167.1 (CONH), 156.6 (C-3), 155.4 (C-4), 149.3 (C-1), 135.2 (C-10a), 134.7 (C-4a), 128.4 (C-1′’), 126.9 (C-3″ to C-5′’), 126.1 (C-2″ and C-6′’), 125.9 (C-2), 124.4 (C-6), 121.9 (C-8), 120.8 (C-7), 118.2 (C-5), 109.6 (C-9a), 72.4 (OCH2), 56.6 (OCH3), 47.7 (C-1′), 22.1 (C-2′)  . HRMS  was dissolved in anhydrous acetone, and NaH  and BrCH2CH2OH  were added. The mixture was kept under reflux and magnetic stirring for 24 h. Then, the organic solvent was evaporated under reduced pressure, and the crude product was dissolved in ethyl acetate. This solution was washed with a 0.5 M NaOH solution (3 × 100 mL) and H2O (2 × 50 mL). The organic layer was dried with anhydrous NaSO4, filtered, and the organic solvent was evaporated under reduced pressure affording a yellow solid.Starting from compound ynthesis . Compounmax (cm−1) (KBr): 3423.2, 1602.0, 1467.0, 1290.5, 1224.5, 1068.1, 905.5, 823.5, 760.4. 1H NMR  δ: 8.33 , 8.12 , 7.84 , 7.53 , 7.39 , 7.03 , 4.33 , 4.05 , 4.03 , 3.90 . 13C NMR  δ: 175.3 (C-9), 157.5 (C-3), 155.5 (C-4), 150.0 (C-1), 135.2 (C-10a), 135.1 (C-4a), 125.8 (C-2), 124.2 (C-6), 121.4 (C-8), 120.8 (C-7), 118.2 (C-5), 115.9 (C-8a), 109.7 (C-9a), 75.1 (OCH2), 60.4 (CH2OH), 56.5 (OCH3)  . HRMS (E87.09042 .S)-15( or R)-15-16 and (R)-16 was carried out using the same method as described, previously, for 2′-[bis(oxy)]diacetate (6), adjusting the amount of K2CO3 and BrCH2COOCH3 according to the number of hydroxyl groups of the chemical substrate and the stoichiometry of the reaction oxy}acetate [S)-16(]: Yield: 80%. m.p.: > 330 °C. [α]D25 °C + 686.0 (c = 0.86 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 1.63) ((k: 1.63) . R)-2-{[9-oxo-7-((1-Phenylethyl)carbamoyl)-9H-xanthen-3-yl]oxy}acetate [R)-16(]: Yield: 75%. m.p.: > 330 °C. [α]D25 °C − 686.0 (c = 0.86 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 0.52) ((k: 0.52) . max (cm−1) (KBr): 3431.7, 3295.6, 1759.3, 1648.4, 1617.9, 1540.7, 1479.7, 1348.7, 1213.5, 1067.7, 828.6, 781.6. 1H NMR  δ: 9.20 , 8.76 , 8.33 , 8.15 , 7.71 , 7.45–7.26 , 7.23 , 5.21 , 5.07 , 3.74 , 1.52 . 13C NMR  δ: 174.9 (C-9), 168.6 (CONH), 164.1 (COOCH3), 163.4 (C-6), 157.3 (C-10a), 157.2 (C-4a), 144.8 (C-1″), 134.1 (C-3), 130.4 (C-2), 128.3 (C-3″ to C-5″), 126.7 (C-8), 126.1 (C-2″ and C-6″), 125.5 (C-1), 118.2 (C-8a), 114.1 (C-7), 101.8 (C-5), 65.1 (OCH2), 52.1 (OCH3), 48.7 (C-1′), 22.2 (C-2′)  . HRMS (E32.14410 .S)-16( and (R)-16 were hydrolysed to the acetic acid derivatives S)-17( and (R)-17 through the method previously described for compound 7 oxy}acetic acid [S)-17(]: Yield: 51%. m.p.: > 330 °C. [α]D25 °C + 450.0 (c = 0.75 × 10−3 gmL−1 in CH3OH). e.r: > 98% (k: 0.56) ((k: 0.56) .R)-2-{[9-oxo-7-((1-phenylethyl)carbamoyl)-9H-xanthen-3-yl]oxy}acetic acid [R)-17(]: Yield: 71%. m.p.: > 330 °C. [α]D25 °C − 450.0 (c = 0.75 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 0.38) ((k: 0.38) .max (cm−1) (KBr): 3306.9, 3082.7, 2977.7, 1718.6, 1664.0, 1619.8, 1552.4, 1480.6, 1236.6, 1065.8, 830.9, 779.2. 1H NMR  δ: 9.19 , 8.74 , 8.31 , 8.14 , 7.72 , 7.44–7.24 , 7.17 , 7.12 , 5.21 , 4.94 , 1.52 . 13C NMR  δ: 174.8 (C-9), 165.4 (CONH), 164.1 (COOH), 163.6 (C-6), 157.3 (C-10a), 157.2 (C-4a), 144.8 (C-1″), 134.0 (C-3), 130.3 (C-2), 128.2 (C-3″ to C-5″), 126.6 (C-8), 126.1 (C-2″ and C-6″), 125.4 (C-1), 118.1 (C-8a), 114.0 (C-7), 101.6 (C-5), 65.0 (OCH2), 48.7 (C-1′), 22.1 (C-2′)  . HRMS -18-18 were synthetized according to the method previously described for compound 8 -2-oxoethoxy}-9-oxo-N-[(S)-1-phenylethyl]-9H-xanthene-2-carboxamide : Yield: 65%. m.p.: > 330 °C. [α]D25 °C − 211.3 (c = 0.71 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 2.58) (6-{2-[((k: 2.58) . S)-2-Hydroxy-1-phenylethyl)amino]-2-oxoethoxy}-9-oxo-N-[(R)-1-phenylethyl]-9H-xanthene-2-carboxamide : Yield: 63%. m.p.: > 330 °C. [α]D25˚C + 211.0 (c = 0.71 × 10−3 gmL−1 in CH3OH). e.r: > 99% (k: 0.47) (6-{2-+ calcd. for [C32H29N2O6]: 537.20256; found 537.20142 (IR ν2 (C-2′) . HRMS -Whelk-O1®  from Regis Technologies, Inc.  were carried out in triplicate. The column void time (t0) was considered to be equal to the peak of the solvent front and was taken from each particular run. The e.r. was determined by the relative percentages of the peak areas according to e.r. (%) = 100 × ([R]/([R] + [S]) or 100 × ([S]/([S] + [R]), where [S] and [R] are the area of the peak of each enantiomer [The cLC analyses of the enantiomeric purity were performed on a Jasco model 880-PU Intelligent HPLC pump , equipped with a JASCO model 880-30 solvent mixer involving an 875-UV intelligent UV-vis detector and a 7125 injector  fitted with a 20 μL loop. The data were handled on ChromNAV Chromatography Data System (version 1.19.1) from JASCO Corporation . The chromatographic column used was (antiomer .Escherichia coli (PDB: 2GFP) as the homolog, as described previously [The crystal structures of the AcrB (PDB: 4DX5) , AcrA (Peviously . The topeviously .Staphylococcus aureus American Type Culture Collection (ATCC) 25923, Enterococcus faecalis ATCC 29212 and methicillin and ofloxacin-resistant Staphylococcus aureus 272123 clinical isolate were used. As Gram-negative strains, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, the acrA gene inactivated mutant Salmonella enterica serovar Typhimurium SL1344 (SE03), and clinical isolates of the extended-spectrum β-lactamase producer (ESBL) E. coli SA/2 were investigated in this study.As Gram-positive strains, Chromobacterium violaceum wild type 85 (wt85), capable of producing violacein, a purple pigment, mediated by AHL signal molecule, capable of endogenous QS-signal molecule production (N-hexanoyl-l-HSL), the Tn5 transposase-mutant C. violaceum CV026 (CV026), capable of producing violacein in the presence of AHL, but incapable of producing endogenous QS-signal molecules, serving as a detector of external stimuli provided by Sphingomonas paucimobilis Ezf 10–17 (EZF), a AHL-producing-strain, and Serratia marcescens AS-1, a producer of prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), an orange-red pigment mediated by the AHL-signal molecules, capable of endogenous QS-signal molecule production (N-hexanoyl-l-HSL). These bacteria are Gram-negative [The bacteria used for the QS assay were negative .Candida albicans ATCC 10231, and as filamentous fungi Aspergillus fumigatus ATCC 204305 and a dermatophyte  were used. Cultures were obtained in SDA.As yeast v/v), was used as a solvent for the compounds.The antibacterial activity was assessed by determination of the MIC of the compounds using the microdilution method, in a 96-well plate, according to the Clinical and Laboratory Standard Institute (CLSI) guidelines . The medE. coli SA/2 when the compound was used alone. The antibiotic tested was serially diluted, whereas the concentration of each compound was kept fixed at 64 µg/mL, which were later converted to µM. Antibiotic MICs were determined as described above.The combined effect of the compounds and clinically relevant antimicrobial drugs was evaluated by determining the antibiotic MIC in the presence of each compound. Briefly, the MIC of cefotaxime (CTX)  was determined in the presence of the highest concentration of each compound tested that did not affect bacterial growth of v/v) was included.The susceptibility tests for yeasts were performed based on the Clinical and Laboratory Standards Institute (CLSI) description for the broth microdilution method on the reference document M27A-3 for yeasts  and M38-5–7, 9, 10, 12, and the enantiomeric pairs 11, 17, and 18, and this effect was studied in the strains SE03 and S. aureus 272123. The intracellular accumulation of EB, a known substrate of bacteria efflux pumps, was monitored through real-time fluorimetry. The method used was automated, using a CLARIOstar Plus plate reader . The positive controls were reserpine and CCCP, and were applied at the non-toxic concentration of 25 µM, as well as DMSO, used as solvent, which was applied at 1% v/v. S. aureus 272123 and SE03 were incubated in TSB and LB-B, respectively, at 37 °C until they reached an optical density (OD) = 0.4–0.6 at λ = 600 nm. The culture was centrifuged at 13,000× g for 3 min, and the pellet was washed and resuspended with PBS. The suspension was centrifuged again in the same conditions and resuspended in PBS. The compounds were diluted to 50 µM in a solution 1 µg/mL of EB, and 50 µL of this solution were transferred into a 96-well black microtiter plate . Then, 50 µL of bacterial suspension (OD600 = 0.4–0.6) were added to each well. The plates were placed into the CLARIOstar plate reader, and the fluorescence was monitored at excitation (530 nm) and emission (600 nm) wavelengths every minute for 1 h on a real-time basis. From the real-time data, the activity of the compounds was expressed as the RFI of the last time point (minute 60) of the EB accumulation assay, which was calculated according to the following formula:treated is the relative fluorescence (RF) at the last time point of EB accumulation curve in the presence of the compound, and RFuntreated is the RF at the last time point of the EB accumulation curve of the untreated control, having only the solvent (DMSO) control.The efflux pump inhibition assay was carried out using compounds S,S)-8-8-17-18. The initial inoculum was incubated in TSB overnight, and then diluted to an OD600 of 0.1. Then, the bacterial suspension was added to 96-well microtiter plates and the compounds were added at a concentration of ½ MIC, and for compounds whose MIC was higher than 100 µM, a concentration of 100 µM was used. The final volume in each well was 200 µL. Reserpine was used as the positive control, as it was the same compound used in the efflux pump inhibition assay and it has shown activity in the inhibition of biofilm formation in S. aureus strains [v/v CV solution were added to the wells and incubated for 15 min at room temperature. Then, the CV solution was removed from the wells, and the plates were washed again with tap water, and 200 µL of a 70% ethanolic solution were added to the wells. The biofilm formation was determined by measuring the OD600 using a Multiscan EX ELISA plate reader . The anti-biofilm effect of the compounds was expressed as the percentage (%) of a decrease in biofilm formation.The enantiomeric pair composed by compounds  strains . The plaS. marcescens, for S,S)-8-8, (R)-17-18 were placed on the center of the inoculated line(s) and impregnated with 8 µL of a solution of 10 mM of the compounds. PMZ, previously described as a QS inhibitor, was used as the positive control [The QS inhibitory effect of the compounds was examined on the EZF and the sensor CV026 strains, on the wt85 strain, and on  control . The aga control ,60,75.In this study, a small library of chiral derivatives of xanthones was synthesized, having as chemical model molecules of antimicrobial peptides. Herein, not only the potential of these derivatives as antimicrobial agents were studied but also their usefulness as antimicrobial helpers, capable of acting in resistance mechanisms and possibly as combination therapy with antimicrobials.6 and S)-11-8-8-17-18-8-8-8( also proved successful in inhibiting QS in the tested strains. These results suggest that, in the case of QS, enantioselectivity does not play an important role, rather than the nature of the substituents.The compounds were also investigated for their ability to inhibit mechanisms related to efflux pumps, such as biofilm formation and QS. It was shown that none of the compounds presented higher inhibition of biofilm formation than reserpine, used as a positive control, but compound In conclusion, it can be observed that these compounds show potential for the activities tested. Future studies will focus on the evaluation of the cytotoxicity of the compounds in eukaryotic cells and in deeper studies of their mechanisms of efflux pump inhibition. Docking studies presented suggest that these compounds can interact with the most relevant efflux pumps, but other mechanisms, such as membrane disturbance, which is characteristic of antimicrobial peptides, could also play a role in the obtained results.