We compared our protocol to that used for heart mitochondrial function that is well-established in the literature, and achieved similar results.Direct analysis of isolated mitochondria enables a better understanding of lung dysfunction. Despite well-defined mitochondrial isolation protocols applicable to other tissues, such as the brain, kidney, heart, and liver, a robust and reproductive protocol has not yet been advanced for the lung. We describe a protocol for the isolation of mitochondria from lung tissue aiming for functional analyses of mitochondrial O The assessment of mitochondrial function in organs and tissues is essential for a better understanding of their biochemistry, physiology, and pathophysiology . The eva1.® beaker, PTFE, low form, catalog number: Z322660; Merck, Darmstadt, Germany).Teflon beaker .50 ml Falcon tubes .4.1.5 and 2 ml microfuge tubes .5.® syringe filters, nylon membrane, diameter 25 mm, pore size 0.2 μm, catalog number: CLS431224; Merck, Darmstadt, Germany).Syringe filter .7.® pipette withdraw volume 3.5 ml, catalog number: 747755; Merck, Darmstadt, Germany).Dispenser pipette .Hamilton syringe: 10 and 25 μl .10.Silica glass cuvettes .11.®).96-well white plate, polystyrene, High Bind, white flat-bottom wells, non-sterile, white .96-well black plate, polystyrene, flat bottom, black polystyrene, matrix active group High Bind, non-sterile  acid ; stored at room temperature (RT).14.Adenosine 5′-diphosphate monopotassium salt dihydrate ; stored at −20°C.15.Adenosine 5′-triphosphate (ATP) assay mix  stored at −20°C.16.Amplex™ Red Reagent  stored at −20°C.17.Cyclosporin A  stored at −20°C.18.Ethylene-bis(oxyethylene dinitrilo)tetraacetic acid  stored at RT.19.Glutamic acid potassium  stored at RT.20.Bovine serum albumin  stored at 4°C. Critical: BSA is used to remove (bind) free fatty acids; therefore, use BSA-free fatty acids.21.2, catalog number: C3306, CAS number: 10035-04-8; Sigma-Aldrich, San Luis, MO, United States) stored at RT.Calcium chloride dihydrate (CaCl22.Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone  stored at 4°C.23.L-Ascorbic acid  stored at 4°C.24.2, catalog number: M2393, CAS number: 7791-18-6; Sigma-Aldrich, San Luis, MO, United States) stored at 4°C.Magnesium chloride hexahydrate (MgCl25.L-(-)-Malic acid  stored at RT.26.N-(2-Hydroxyethyl)piperazin-N′-(2-ethanesulfonic acid)]  stored at RT.27.N,N,N,N-Tetramethyl-p-phenylenediamine dihydrochloride  stored at RT.28.Potassium chloride  stored at RT.29.2PO4, catalog number: P5655, CAS number: 7778-77-0; Sigma-Aldrich, San Luis, MO, United States) stored at RT.Potassium dihydrogen phosphate  stored at RT.Sodium phosphate dibasic  stored at RT.32.Sucrose  stored at RT.33.Trizma base  stored at RT.34.Tetramethylrhodamine methyl ester perchlorate  stored at −20°C.1.Isolation buffer in mmol/l: sucrose 250; HEPES 10; EGTA 1, pH 7.4. Dissolve 85.58 g of sucrose, 2.6 g of HEPES, and0.38 g of EGTA in 800 ml of ultrapure water. Adjust pH to 7.4 using 2 mol/l Tris, and bring the solution to 1 L and store at 4°C.2.BSA isolation buffer: Dissolve 400 mg of BSA fat-free in 50 ml isolation buffer.3.2HPO4 374; KH2PO4 191; KCl 139.5; NaN3 15.38. Dissolve 2.655 g of Na2HPO4, 1.3 g of KH2PO4, and 0.52 g of KCl in 50 ml ultrapure water. Add 0.05 g of NaN3 and a few crystals of AgCl to provide a saturated solution. Filtrate the solution and store at 4°C. Caution: NaN3 is highly toxic.Electrolyte solution in mmol/l: Na4.2; 5 KH2PO4;0.02 EGTA; 5 pyruvate/malate, pH 7.4. Add 6.25 ml of 1 mol/l KCl, 1 ml of 500 mmol/l MOPS, 0.1 ml of 1 mol/l MgCl2, 0.25 ml of 1 mol/l KH2PO4, 0.1 ml of 100 mmol/l EGTA, and 1 ml of 250/250 mol/l pyruvate/malate. Adjust pH to 7.4 using 500 mmol/l Tris, and bring the solution to 50 ml using ultrapure water and filtrate it. Store at 4°C.Incubation buffer for respiration pyruvate/malate (IBRP/M) in mmol/l: 125 KCl; 10 MOPS; 5 MgCl5.2; 5 KH2PO4;0.02 EGTA; 5 succinate, pH 7.4. Add 6.25 ml of 1 mol/l KCl, 1 ml of 500 mmol/l MOPS, 0.1 ml of 1 mol/l MgCl2, 0.25 ml of 1 mol/l KH2PO4, 0.1 ml of 100 mmol/l EGTA, and 1 ml of 250 mmol/l succinate. Adjust pH to 7.4 using 0.5 mol/l Tris, and bring the solution to 50 ml using ultrapure water, and filtrate it and store at 4°C.Incubation buffer for respiration succinate (IBRS) in mmol/l: 125 KCl; 10 MOPS; 5 MgCl6.100 mmol/l ADP: Dissolve 427 mg of ADP in 10 ml of ultrapure water. Prepare 100 μl aliquots and store at −20°C.7.500 mmol/l ascorbate: Dissolve 880.65 mg of ascorbic acid in 10 ml of ultrapure water. Prepare 100 μl aliquots and store at −20°C.8.2 in 50 ml of ultrapure water and store at −20°C.10 mmol/l calcium chloride: Dissolve 55.49 mg of CaCl9.10 mmol/l cyclosporin A: Dissolve 12 mg of cyclosporin A in 1 ml of absolute ethanol and store at −20°C.10.0.1 mol/l EGTA stock solution: Dissolve 1.9 g of EGTA in 30 ml of ultrapure water. Adjust pH to 7.4 using 0.5 mol/l Tris and dilute to 50 ml. Store at 4°C.11.10 mmol/l FCCP stock solution: Dissolve 2.5 mg of FCCP in 1 ml of absolute ethanol. Store at −20°C. Dilute the stock solution to 5 μM by adding 5 μl of 10 mmol/l FCCP in 10 ml of absolute ethanol. Prepare 20 μl aliquots and store at −20°C.12.0.25 mol/l Pyruvate/0.25 mol/l malate stock solution: Dissolve 1.38 g of pyruvate and 1.68 g of malate in 30 ml of ultrapure water and adjust pH to 7.4 with 2 mol/l Tris. Dilute to 50 ml and store at 4°C.13.1 mol/l KCl stock solution: Dissolve 18.64 g of KCl in 250 ml of ultrapure water and store at 4°C.14.2PO4 stock solution: Dissolve 6.8 g of KH2PO4 in 30 ml of ultrapure water. Adjust pH to 7.4 using 0.5 mol/l Tris and dilute to 50 ml. Store at 4°C.1 mol/l KH15.2 stock solution: Dissolve 4.7 g of MgCl2 in 50 ml of ultrapure water and store at 4°C.1 mol/l MgCl16.0.5 mol/l MOPS stock solution: Dissolve 10.46 g of MOPS in 30 ml of ultrapure water. Adjust pH to 7.4 using 0.5 mol/l Tris and dilute to 100 ml. Store at 4°C.17.1 mmol/l rotenone stock solution: Dissolve 3.9 mg of rotenone in 10 ml of absolute ethanol. Dilute the stock solution to 500 μmol/l by adding 5 ml of 1 mM rotenone in 5 ml of absolute ethanol. Prepare 200-μl aliquots and store at −20°C . Rotenone is light-sensitive. The stock solution should be protected from direct light.18.0.25 mol/l succinate stock solution: Dissolve 2.02 g of succinate in 30 ml of ultrapure water and adjust pH to 7.4 with 2 mol/l Tris. Dilute to 50 ml and store at 4°C.19.150 mmol/l TMPD: Dissolve 49.3 mg of TMPD in 2 ml DMSO. Prepare 10-μl aliquots and store at −20°C.20.2 mol/l Tris: Dissolve 121.14 g of Tris in 500 ml of ultrapure water. Dilute to 0.5 mol/l by adding 250 ml 2 mol/l Tris in 750 ml ultrapure water and store at RT.21.5 mmol/l TMRM stock solution: Dissolve 5 mg of TMRM in 2 ml of DMSO. Store at −20°C.1.instrumentos cirúrgicos, surgery scissors straight 12 cm, code: 321. Catalog number: 10304850053).Surgery scissors .3.®, catalog number: 3725000; Merck, Darmstadt, Germany).Tissue homogenizer .5.® M3 .Spectrofluorimeter SpectraMax6.Centrifuges and rotors: Precool centrifuges and rotors to 4°C.7.Oxygraph chamber: Adjust the temperature of the water bath to 37°C. Calibrate the Clarke-type oxygen electrode. Procedures may vary from instrument to instrument. Follow the manual for the oxygen electrode and chamber you are using .8.Spectrophotometer: Adjust the temperature of the cuvette block to 37°C.1.Strathkelvin 782 2-channel Oxygen System version 1.0 .2.® Pro Software .SoftMax3.GraphPad Prism 8.4.3 .CD-1 mice (25–30 g BW) were used. The animal study was reviewed and approved by our institutional ethics committee on the use of animals  and followed the guidelines of the Brazilian National Council for Animal Experimentation Control, Ministry of Science, Technology, and Innovation (CONCEA/MCTI), and the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health .The experimental protocol must be available after lung mitochondrial isolation, because mitochondria are viable for about 4 h only .en bloc and immediately placed in a tube containing an ice isolation buffer at 4°C (see section “Recipes”) to remove excess blood.The mice were euthanized and underwent a bilateral thoracotomy. The lungs were carefully removed The following steps are critical for the isolation of mitochondria . Mistakes during mitochondria isolation are irreversible and can spoil the running experiment. All processes must be performed on ice. Centrifugation steps at 4°C and buffers should be precooled during processing. It is important to work fast to avoid delays in tissue preparation .a.Remove the adipose tissue and all large vessels using scissors.b.Mince the tissue into 1–2 g fragments and transfer each one into a Teflon beaker with 10 ml BSA isolation buffer on ice.c.Remove all remaining fat. The tissue must be thoroughly minced, since the size of the sample directly affects the subsequent homogenization step and eventually the yield of mitochondria . Ensure the removal of all fats that also affect the yield of mitochondria.d.Split the minced tissue from one Teflon beaker into two 14-ml round-bottom tubes. The tube should not contain more than 2 ml tissue volume. Whenever necessary, use more 14-ml round-bottom tubes.e.Wash the minced tissue samples: fill the 14-ml round-bottom tubes with 10 ml BSA isolation buffer, let the tissue sink, remove the buffer, and repeat tissue washing until the buffer is clear (the minced tissue would then contain no blood). Usually, four or five washings are enough to obtain a clean BSA isolation buffer. Hence, fill the 14-ml round-bottom tubes up to 6 ml with isolation buffer. The optimal tissue/buffer ratio is 1:3 or less. After mincing the tissue, part of it precipitates and some pieces float because of the air in the air spaces . Be careful in removing the blood during washing to avoid tissue loss.f.Homogenize the samples with the tissue homogenizer (Ultra-Turrax) using two 10-s treatments at a shaft rotation rate of 6,500 × g each. Perform the homogenization on ice with slight movements of the centrifuge tube. Wait for 10 s between the homogenization steps to avoid heating of the homogenizer and the samples, and to avoid foaming .g.Collect the samples and transfer them to a tissue glass Potter-Elvehjem homogenizer. Homogenize the samples, and stroke the suspension about 30–40 times. This procedure can compromise mitochondrial integrity if not done carefully . It Is recommended to precool the glassware in an ice bath 5–10 min before starting the procedure. Attention: The use of proteases, e.g., nargase, during mitochondrial isolation, commonly performed in other tissues such as the heart, ruins the whole process.h.Centrifuge the homogenate at 700 × g for 10 min at 4°C.i.Collect the supernatant in 2-ml microfuge tubes and discard the pellets. Centrifuge the supernatant at 12,300 × g for 10 min at 4°C.j.Discard the supernatant and resuspend the pellet in 0.5 ml of ice-cold isolation buffer by gentle pipetting, and collect the mitochondrial suspensions in 2-ml microfuge tubes. Avoid the formation of foam during the resuspension process .k.Centrifuge the supernatant in ice-cold isolation buffer at 10,300 × g for 10 min at 4°C.l.Pool all the mitochondrial suspensions in one 2-ml microfuge tube and repeat the previous step.m.Resuspend the resulting pellet in 100–200 μl isolation buffer and store it on ice. Resuspend the pellets carefully by gentle pipetting to obtain a uniform suspension without any visible clump .n.Measure mitochondrial concentration using the Lowry or BSA method.At this point, the mitochondria are ready to be used in experiments to explore their function. Use the preparation within 4 h for better functional responses. Store the mitochondrial suspension on ice.Note: The isolated mitochondria by this protocol can be used in different oximeter equipment, and can be analyzed with different software and methodologies.In each experiment, use 200 μg of protein per ml for good acquisition data. The oxygen consumption gives information about the electron transport chain and the oxidative phosphorylation of the mitochondria. By the addition of substrates and inhibitors, or by uncoupling oxidative phosphorylation, it is possible to modulate the rate of oxygen consumption and gain further insight into the activity of each complex of the electron transport chain.At this point, mitochondrial complexes I , II (state 3), and IV respiration with subsequent uncoupling of oxidative phosphorylation were measured in a two-chamber respirometer. With two different chambers, it is possible to measure two different experimental groups at the same time, observing results in mitochondrial function in parallel . Moreover, the respiration of complex I and complex II can be available in parallel using two different chambers.a.+) to measure the state 1 respiration of complex I and to add IBRS plus 2 μmol/l rotenone to the other chamber to measure complex II respiration. Make sure that the magnetic stirrer moves constantly. Rotenone is sticky and inhibits complex I respiration . Therefore, we suggest washing each chamber that received rotenone with a cardiac or liver tissue homogenate to assist the removal of rotenone. Additionally, we recommend washing the chamber three times with 70° alcohol, followed by three washes with EDTA 100 mmol/l. Finally, wash 10 times with MilliQ water.Add 0.5 ml of IBRP/M buffer to the chambers. One can opt to add IBRP/M without pyruvate/malate . Wait for 1–5 min to obtain a stable baseline. A maximal drift of ±10 nmol Oor 3 min .d.Add 4 μl of 100 mmol/l ADP to obtain a final concentration of 400 μmol/l using a Hamilton syringe and record oxygen concentration for 3 min .e.The decrease in oxygen concentration speeds up, caused by stimulating mitochondrial respiration with ADP (state 3). ADP-stimulated respiration should be faster than baseline respiration, reflecting good coupling of mitochondria . The respiration could slow down and return to a rate comparable to that of the baseline respiratory state, as result of the conversion of all added ADP and phosphate into ATP.f.Add simultaneously 2 μl of 150 mmol/l TMPD and 6 μl of 500 mmol/l ascorbate to obtain final concentrations of 300 and 3 mmol/l, respectively. Record oxygen concentration for 1 min. The oxygen concentration will decrease faster than with ADP stimulation. TMPD is an electron donor to complex VI, which is readily reduced by ascorbate and oxidized by cytochrome C .g.Add 6 μl of 5 μmol/l FCCP to obtain a final concentration of 30 nmol/l. Record oxygen concentration for 1 min. The oxygen concentration will decrease further. FCCP is an uncoupling agent, which turns the mitochondrial membrane permeable to protons and, therefore, eliminates the chemiosmotic gradient. As a result, ATP synthesis is disrupted .h.Stop recording.i.Calculate mitochondrial oxygen consumption using the software Analysis  or similar. Calculate baseline oxygen consumption 75 s after the addition of mitochondria. Calculate state 2 complex I oxygen consumption 75 s after the addition of pyruvate/malate. Determine ADP-stimulated respiration 30 s after the addition of ADP. Determine complex VI respiration 30 s after the addition of ascorbate/TMPD. Calculate maximal uncoupled respiration rate 30 s after the addition of FCCP.® M3; Molecular Devices, San Jose, CA, United States) at 560-nm emission.In the respiration chamber, repeat all the steps up to adding 4 μl of 100 mmol/l ADP to obtain a final concentration of 400 μmol/l. Then, record the ADP-stimulated respiration for 3 min. Thereafter, the incubation buffer containing mitochondria should be transferred to an Eppendorf tube and immediately supplemented with ATP assay mix  diluted to 1:5 (incubation buffer containing mitochondria: ATP assay mix). Mitochondrial ATP production was determined immediately after each respiration measurement and compared with ATP standards using a 96-well white plate in a spectrofluorometer  at 540-nm emission and 580-nm extinction  was used to determine mitochondrial ROS concentration. Amplex Red reacts in 1:1 stoichiometry with peroxide in the presence of horseradish peroxidase (HRP) and produces highly fluorescent 95% resorufin. The incubation buffer containing mitochondria should be transferred to an Eppendorf tube and immediately supplemented with 50 μmol/l Amplex UltraRed Reagent  and 2 U/ml Pierce™ horseradish peroxidase . The supernatant was collected after 20 min of incubation in the dark. Mitochondrial ROS concentration was determined and compared with Htinction .The integrity of the mitochondrial membrane was assessed by osmotically induced volume changes of the mitochondria and spectrophotometric determination of the apparent absorption of the suspension at 540 nm. A mitochondrial suspension (200 mg/ml) was added to the respiration medium in the absence of respiratory substrates, at 37°C, and under constant stirring. Mitochondrial swelling was stimulated with 1 μl of calcium chloride at 20 μmol/l to reach 100 nmol/l in 200 μl of mitochondrial suspension. Swelling was expressed as percentage of the absorption of the solution containing mitochondria in the presence of cyclosporin A 10 μmol/l  in relation to that absorbed after the addition of FCCP 1 μmol/l .For ΔΨm determination, the probe tetramethylrhodamine methyl ester  was added to the respiration solution containing 200 mg/ml of mitochondria and incubated for 1 h at 4°C before the experiment. ΔΨm was estimated by the fluorescence emitted by TMRM under 580-nm excitation. ΔΨm was expressed as the percentage of fluorescence emitted by TMRM-labeled mitochondria in the presence of cyclosporin A , relative to that emitted after the addition of FCCP to fully depolarize the mitochondria .2-). However, other reactive oxygen species, such as hydroperoxyl radical (HO2) and hydrogen peroxide (H2O2), might occur spontaneously  or under the action of antioxidant enzymes . The site of initial leakage is often considered to be a semiquinone radical (QH) or reduced flavin (FMN and FAD) (A–B). To calculate the fraction of electrons that leaked out of the respiratory chain, the rate of H2O2 formation (see section E) is divided by the rate of mitochondrial O2 consumption (see section C). H2O2 production and oxygen consumption rates must be expressed using the same units and correspond to the same respiratory state (C–E). The ATP/ROS reason should be measured to determine the formation of ROS linked to O2 consumption. Thus, we were able to determine the electron leakage inherent to ROS production  was used. The significance of observed differences in mitochondrial oxygen consumption and functions was evaluated by the parametric one-Way ANOVA test followed by Tukey’s multiple comparisons test. In all cases, The differences between the present protocol and previous protocols are shown in 2/min/mg protein) than in heart mitochondria  and in lung mitochondria-200 μg , which did not differ between them (p = 0.7).2/min/mg protein) than in heart mitochondria  and in lung mitochondria-200 μg , which did not differ between them (p = 0.99).As shown in 2/min/mg protein) than in heart mitochondria  and in lung mitochondria-200 μg , which did not differ, p = 0.99, as presented in The mitochondrial oxygen consumption by complex I under state 3 was smaller in lung mitochondria-50 μg (26.75 ± 8.2 nmol O2/min/mg protein) than in heart mitochondria-50 μg , and in lung mitochondria-200 μg . However, the increase in oxygen consumption by lung mitochondria-200 μg did not reach the level of the heart mitochondria-50 μg (p = 0.009), as shown in The mitochondrial oxygen consumption by complex II under state 3 was smaller in lung mitochondria-50 μg (35.54 ± 11 nmol O2/min/mg protein) than in heart mitochondria-50 μg  and lung mitochondria-200 μg , which did not differ between them (p = 0.19). The mitochondrial oxygen consumption by maximal oxygen uptake of uncoupled mitochondria was similar in heart mitochondria-50 μg and lung mitochondria-50 μg . Lung mitochondria-200 μg showed similar respiration to heart mitochondria-50 μg  and p = 0.5 vs. lung mitochondria-50 μg) (a-50 μg) .p = 0.003) and lung mitochondria-200 μg , which was similar (p = 0.87), as displayed in Mitochondrial ROS production by lung mitochondria-50 μg (21.87 ± 9.6 nmol/loaded protein) was lower than that by heart mitochondria-50 μg  and lung mitochondria-200 μg , which was similar (p = 0.075).p = 0.3), as depicted in The mitochondrial swelling of lung mitochondria-50 μg (68.5 ± 19.7% maximum), heart mitochondria-50 μg (63.9 ± 13.8% maximum), and lung mitochondria-200 μg (81.84 ± 15.35% maximum) did not differ among them (p = 0.52) , heart mitochondria-50 μg (59.46 ± 19.2% maximum), and lung mitochondria-200 μg (77.51 ± 14.1% maximum) did not differ among them ( = 0.52) .2O2 production/O2 consumption), heart mitochondria-50 μg (0.84 ± 0.31 H2O2 production/O2 consumption), and lung mitochondria-200 μg (0.97 ± 0.22 H2O2 production/O2 consumption) was similar (p = 0.48).2O2 production/ATP formation), heart mitochondria-50 μg (1.07 ± 0.42 H2O2 production/ATP formation), and lung mitochondria-200 μg (1.39 ± 0.35 H2O2 production/ATP formation) did not differ (p = 0.54), as depicted in The mitochondrial ATP/ROS ratio of lung mitochondria-50 μg  comprised mitochondria isolated from the heart carrying 50 μg of protein per experiment. Heart mitochondria are isolated by means of a well-established broadly used protocol , 2021. TThe lung is an extremely complex organ with regard to the heterogeneity of cells. Our method does not contemplate analyzing all 40 subtypes of cells found in the lung. However, this heterogeneity is an intrinsic part of the lung, and all methods of mitochondria isolation, for most diverse tissues, contemplate entire organ isolation, because different cells form a syncytium for the organ to work, e.g., the heart , kidney Based on the method briefly described by The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the UFRJ–local Institutional Animal Care and Use Committee (015/17). Written informed consent was obtained from the owners for the participation of their animals in this study.LM, WZ, and JN: conception and design, data acquisition, analysis and interpretation of data, drafting or revising the article, and contribution with reagents. DC, DO, and JC-d-A: data acquisition, and analysis and interpretation of data. LM was the principal investigator. All the authors discussed the results, commented on the manuscript, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The sulfamide derivative has a notably long murine microsomal half-life (t1/2 > 150 min), low intrinsic liver clearance and total plasma clearance rates , and high relative bioavailability (F = 59%). Kinetics are somewhat similar for 11-aza-artemisinin , although bioavailability is lower (F = 14%). In contrast, artemether is rapidly metabolized to dihydroartemisinin (DHA) (t1/2 = 17.4 min) and eliminated  and has low oral bioavailability (F) of 2%. While artemisone displays low t1/2 of <10 min and high CLint of 302.1, it displays a low CLtot of 42.3 ml/min/kg and moderate bioavailability (F) of 32%. Its active metabolite M1 displays a much-improved t1/2 of >150 min and a reduced CLint of 37.4 ml/min/kg. Artemiside has t1/2 of 12.4 min, CLint of 673.9, and CLtot of 129.7 ml/kg/min, likely a reflection of its surprisingly rapid metabolism to artemisone, reported here for the first time. DHA is not formed from any amino-artemisinin. Overall, the efficacy and PK data strongly support the development of selected amino-artemisinins as components of new TACTs.As artemisinin combination therapies (ACTs) are compromised by resistance, we are evaluating triple combination therapies (TACTs) comprising an amino-artemisinin, a redox drug, and a third drug with a different mode of action. Thus, here we briefly review efficacy data on artemisone, artemiside, other amino-artemisinins, and 11-aza-artemisinin and conduct absorption, distribution, and metabolism and excretion (ADME) profiling  Plasmodium falciparum to the artemisinin component   comprising one of dihydroartemisinin (DHA) 2, artemether 3, or artesunate 4  with pipomponent –3. Althoonent –3–. Resista PfK13) –11. It iiescence , 13. The –11. It iescence –17. The escence –, 19, or escence –, 17. In escence –. Howeverescence – or pharmescence –, 23. Alsescence –, 25. Theescence ––28. Irrescence ––. Neverthscence ––.The artemisinins in the current ACTs  have bee31–34–37–41–p-fluorophenyl)amino derivative 8 (p-fluorophenylpiperazine) urea derivatives 9  and PL08-009 (5C) carrying the PfKI3 C580Y mutation (P. berghei (Pb) sporozoites , 5251, 556–vative 8 , the 10-atives 9  and 10  activities ranging from 0.85 to 1.75 nM against asexual blood stages of CQ-sensitive PfNF54 and multidrug-resistant K1 and W2 strains. The sulfamide, arylamine, and phenylurea are slightly less active against all strains (1.3 to 4.7 nM), and 11-aza-artemisinin is the least active (6.02 to 10.48 nM) (Table S2) (50] 2.4 to 2.9 μM) are more toxic than artemiside, artemisone, and the arylurea (204 to >271 μM) (Table S1)  against early-stage gametocytes (EG > 90% stage I to III) ranging from 1.94 nM for artemisone to 83 nM for the phenylurea. Against late-stage gametocytes (LG > 90% stage IV and V), the sulfamide is the least active (IC50 419.4 nM), with activities of the other compounds ranging from 1.5 to 42.4 nM. Thus, the latter are appreciably more active than methylene blue, a known gametocytocidal agent (50 of 95 nM and LG of 143 nM (Table S3). These activities thus portend utility of the amino-artemisinins as transmission-blocking agents (50 of 170.4 and LG of 166.1 nM (Table S4) ; in comparison, artemether has an IC50 of >104 nM  are equipotent against clone ARC08-22 (4G) (1.62 to 2.43 nM) and more active against the PL08-009 (5C) clone (0.27 to 0.29 nM). The phenylurea is also active against the ARC08-22 (4G) clone. DHA elicits activities of 6.41 to 6.68 nM against these clones (Table S6)  isolates transfected to strains CamWTC580Y (C580Y K13 mutation), Cam3.IIC580Y (C580Y K13), and Cam3.IIRev (wild-type K13 sequence), wherein it essentially retains baseline activities across all strains, as reflected in a mean IC50 2.4 nM  but less so than artemisone (IC50 0.7 to 1.1 nM)  . Cytotoxable S1) , 48. Theal agent , with EGg agents , 48. 11-able S4) . Against 1.1 nM) . However 1.1 nM) . Work isin vitro. However, expression of therapeutic efficacy depends not only on biological activity but also on pharmacokinetic (PK) properties. This becomes important for the envisaged drug combinations  properties and PK measurement in a murine model of the compounds of Overall, artemiside, artemisone, and the arylurea are the frontrunners in terms of global efficacies and relatively low toxicities inations . We repoHE for mouse, rat, and human liver microsomes  of 3.98   of 0.02.The artemisinins presented moderate plasma protein binding as reflected in the unbound fraction ranging from 0.13 to 0.25. The one exception is provided by the arylurea 10, which had the highest plasma protein binding as reflected in the fraction unbound  and precision (percentage of coefficient of variation [CV]) statistics of the low-, medium-, and high-quality controls for artemether, artemiside, the phenylurea, arylurea, and 11-aza-artemisinin were between 72.2 to 125.9% and 0.2 to 11.8%, respectively, and were 71.2% to 111.1% and 0.7 to 14.5% for artemisone and sulfamide, respectively, during sample analysis. Tafenoquine is an 8-aminoquinoline and is prescribed for prophylaxis and treatment of P. vivax infections   and oralfections  are inclfections  of the lThe plasma concentration-time curves derived from the PK data are shown in Fig. 3 to in vitro ADME data  for artemisone. The discrepancy arises because the ex vivo bioassay screen measures total activity of artemisone plus the active metabolite M1 and the hydroxylated counterparts in monkey or human plasma.Artemiside displayed a short half-life (1.8 h) as well as a high clearance rate (673.9 ml/min/kg) and a low bioavailability of 1%. Notably, we record here for the first time that it was relatively rapidly metabolized to artemisone and subsequently into the unsaturated metabolite M1 . ArtemisDME data . DespiteDME data . It was DME data  and possDME data , 54. Thu2 to 3 h . This wain vitro ADME data which suggest that it is notably stable with a low intrinsic clearance rate  following i.v. or p.o. dosing but revealed promising exposure levels with a bioavailability of 59% . It exhince rate . The phence rate  had a ponce rate , differince rate , presentImportantly, DHA was not detected as a metabolite of either artemiside or artemisone , thThe data overall highlight the problems of metabolic stability, poor solubility, and short half-lives of the artemisinins at large. In addition, a comparison with the far more stable and better absorbed tafenoquine emphasizes the lack of drug-likeness and difficulties in applying drug-design precepts to artemisinins , 75. HowThe inability to measure the kinetic solubility of DHA or to assess the extent of plasma protein binding because of decomposition reflects its instability , 46, 51.Leishmania donovani . Also, the AUC of artemisone generated in situ following oral dosing of artemiside was 9-fold lower than the exposure level achieved upon oral dosing of artemisone itself   . From a tabolism , 80, desCmax and a distinctly higher bioavailability   . The aryable S1) , and botable S1) . The comx values .in vitro  in order to inhibit the malaria parasite at different stages of its life cycle and thus relieve clinical symptoms, avoid relapse, and block transmission . The amiene blue , 48. Theetocytes . The higin vitro ADME and in vivo PK properties that are similar or improved compared to those of DHA and artemether. Although artemisone is readily metabolized to M1 as reflected in its low elimination half-life, it displays a greater bioavailability  in accordance with the South African National Standards SANS 10386:2008) for the Care and Use of Animals for Scientific Purposes  and guid:2008 forPreparation of the amino-artemisinins 5 to 10 and 11-aza-artemisinin 11 and assessment of purity are described elsewhere , 52, 62.2PO4 and K2HPO4 were purchased from Merck . Analytical-grade acetonitrile was purchased from Anatech . Analytical-grade dimethyl sulfoxide (DMSO), formic acid, carbamazepine, propranolol hydrochloride, warfarin, procaine hydrochloride, and vinpocetine were obtained from Sigma-Aldrich . NADPH was purchased from Sigma-Aldrich . Polyethylene glycol (PEG), polypropylene glycol (PPG), and hydroxypropylmethylcellulose (HPMC) were purchased from Sigma-Aldrich . Human, rat, and mouse liver microsomes were obtained from Xenotech . All other reagents were of bioanalytical grade. Water was purified via a Milli-Q purification system .Human whole blood and human plasma were obtained from the Western Province blood transfusion services, Cape Town, South Africa. KH2PO4 (0.2 g), K2HPO4 (0.115 g), NaCl (0.8 g), and KCl (0.2 mg) in purified water (100 ml). The pH was measured and adjusted using 0.05 M HCl or 0.05 M NaOH to pH 7.4. The pH 2 solution was prepared by adding HCl  to purified water (50 ml). FaSSIF buffer at pH 6.5 was prepared by adding KH2PO4 (2.042 g) and KCl (3.728 g) to purified water (450 ml) and adjusting the pH to 6.5 using 0.05 M HCl or 0.05 M NaOH as required. Simulated intestinal fluid (SIF)  powder (5.6 mg) was then added to the buffer (25 ml) to complete the process.For kinetic solubility assays, three solutions with differing pH were prepared. Phosphate-buffered saline (PBS) was prepared by mixing KH3COOH (5.72 ml), H3PO4 (6.82 ml), and H3BO3 (6.183 g) in purified water (1 liter) and buffer B comprising NaOH (20 g) in purified water (1 liter).For the liquid-liquid extraction of plasma samples, a universal buffer containing the internal standard at pH 8 was utilized. The universal buffer consists of a 1:1 mixture of buffer A comprising CH(i) Kinetic solubility. Duplicate stock solutions of each compound at a concentration of 10 mM in DMSO were prepared in a 96-well plate in PBS at pH 7.4, water at pH 2, and FaSSIF buffer at pH 6.5 to a final concentration of 200 μM  Metabolic stability in liver microsomes. The metabolic stability assay was performed in duplicate using 96-deepwell plates. Each compound (0.4 mM) was incubated individually in mouse, rat, and pooled human liver microsomes (0.4 mg/ml) at 37°C in the presence and absence of the cofactor NADPH (1 mM). An aliquot was taken at 0, 5, 10, 30, and 60 min, and the reaction was quenched by adding 300 μl of ice-cold acetonitrile containing the internal standard carbamazepine (0.0236 μg/ml)  . The tesquations .Equation(iii) Plasma protein binding. Plasma protein binding was measured using an ultracentrifugation method. The assay was performed using a 96-well microtiter plate with pooled human plasma spiked with the test compound (10 μM). An aliquot was removed and quenched using ice-cold acetonitrile containing the internal standard carbamazepine (0.0236 μg/ml) and placed in the freezer. This served as the total concentration sample. After preincubation at 37°C for 1 h, duplicate aliquots of the spiked plasma were transferred to ultracentrifugation tubes and ultracentrifuged for 4 h . Analyte concentration of all compounds and sample types were determined by means of LC-MS/MS as described in “Sample analysis” below.(i) Animals. Healthy male C57/BL6 mice each weighing approximately 25 g were used. The mice maintained at the University of Cape Town animal facility were housed in 27 by 21 by 18 cm cages, under controlled environmental conditions at 22 ± 2°C and a 12-h light/dark cycle. Humidity was monitored and ranged between 55 and 70%. Food and water were available ad libitum. Mice were acclimatized to their environment for 7 days before experiments were initiated.(ii) Oral drug administration. Each of artemether, the amino-artemisinins 5 to 10, and 11-aza-artemisinin 11 (12.5 mg) was dissolved in 0.2 ml DMSO, to which 1.8 ml 0.5% (wt/vol) HPMC in water was then added. Compounds were administered orally via gavage at a dose of 50 mg/kg (n = 3). Dosing volumes were calculated based on the weight of each mouse in the respective group. The dosing volumes ranged between 160 μl and 200 μl, ensuring that the dose remained at 50 mg/kg. Blood samples for amino-artemisinins 5 to 8 and 10 were collected via tail-bleeding predose and at 0.08, 0.25, 0.5, 1, 2, and 4 h. Blood samples for the phenylurea 9 and 11-aza-artemisinin 11 were collected at 0.08, 0.5, 1, 2, 4, 8, and 24 h postdose. Samples were placed in 0.5 ml lithium heparin micro vials to prevent blood coagulation. Artemether and the amino-artemisinins 5 to 8 and 10 were expected to display the characteristic short half-lives of the artemisinins, and therefore it was more important to map the initial exposure over 4 h. The phenylurea 9 and the 11-aza-artemisinin 11 were predicted to have longer half-lives and were sampled over a greater time period. The samples were immediately centrifuged at 5,590 × g for 5 min and the plasma was transferred to new microvials. The plasma samples were stored at −80°C.(iii) Intravenous drug administration. For the intravenous formulations, each of artemether, the amino-artemisinins 5 to 10, and 11-aza-artemisinin 11 (2.5 mg) was dissolved in 1 ml DMSO/PEG/PPG . DMSO was added first, followed by PEG and then PPG. The resulting solutions in the mixture of DMSO, PEG, and PPG  were administered intravenously at a dose of 10 mg/kg (n = 3) via injection into the dorsal penile vein. Mice were anesthetized with an intraperitoneal injection of a saline ketamine/xylazine solution. This consisted of 1 ml ketamine (100 mg/ml), 0.5 ml xylazine (20 mg/ml), and 8.5 ml PBS solution to make a total volume of 10 ml. Dosage of anesthetic preparation for mice was 0.8 ml/10 g body weight; this amounts to a dose of 80 mg/kg ketamine and 10 mg/kg xylazine. The total volume of administration was dose adjusted to 10 mg/kg with an approximate volume of 80 μl, according to the weight of each animal on the morning of dosing. Blood samples for the amino-artemisinins 5 to 8 and 10 were collected via tail-bleeding predose and at 0.08, 0.25, 0.5, 1, 2, and 4 h, and blood samples for the phenylurea 9 and 11-aza-artemisinin 11 were collected at 0.08, 0.5, 1, 2, 4, 8, and 24 h postdose. Samples were all placed in 0.5 ml lithium heparin microvials to prevent blood coagulation. The samples were immediately centrifuged at 5,590 × g for 5 min and the plasma was transferred to new microvials. The plasma samples were stored at −80°C.Concentrations of the kinetic solubility, metabolic stability, plasma stability, plasma protein binding samples, and plasma samples from murine studies were determined using quantitative LC-MS/MS assays.Sample extraction. For sample extraction from the kinetic solubility, metabolic stability, plasma stability, and plasma protein binding studies, each compound was extracted from 200 μl of acetonitrile by adding 350 μl ethyl acetate, which contained the internal standard carbamazepine, and vortexing for 1 min followed by centrifugation at 5,590 × g for 5 min. The organic layer was then transferred and evaporated under a gentle stream of nitrogen at room temperature for 30 min. The dried samples were reconstituted in mobile phase, and a 5 μl sample was injected for LC-MS/MS analysis. The only exception was the arylurea 10, for which no liquid-liquid extraction was conducted; instead, the 200 μl acetonitrile solution was evaporated under a gentle stream of nitrogen at room temperature. The dried sample was reconstituted in the mobile phase, and a 5 μl sample was injected for LC-MS/MS analysis. The plasma samples for each compound from the in vivo studies were extracted by mixing 350 μl ethyl acetate with 15 μl of plasma sample and 35 μl of universal buffer containing internal standard at pH 8. The samples were vortexed for 1 min and centrifuged for 5 min, and the organic layer was then transferred and evaporated under a gentle stream of nitrogen at room temperature for 30 min. The dried samples were each reconstituted in the mobile phase, and a 5 μl sample was injected for LC-MS/MS analysis.LC-MS/MS analysis. For all the artemisinins except the arylurea 10, gradient chromatography was performed on a Phenomenex  Luna PFP(2) analytical column  and an AB Sciex API 5500 mass spectrometer which was operated at unit resolution in the multiple reaction monitoring (MRM) mode. The HPLC method used was a gradient method with 5 mM ammonium acetate and 0.1% acetic acid in water as the aqueous phase and a mixture of acetonitrile and methanol (1:1 vol/vol) containing 5% of the aqueous phase as the organic phase , volume of distribution (V), and oral bioavailability (F) could be determined using the noncompartmental analysis software PK Solutions version 2.0, Summit Research Services, Montrose, USA. The area under the plasma concentration curve up to the last time point AUC0–last was calculated by using the trapezoidal rule (dal rule .


Zea mays seedlings to 0.5 mM BOA-OH elicits root zone-specific reactions by the formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs, and no visible defense reaction at the root tip. We studied BOA-6-OH- short-term effects on membrane lipids and fatty acids in maize root tips in comparison to the benzoxazinone-free species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10–30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovered within one hr. Disturbances in membrane phospholipid contents were balanced in both species within 30–60 min. Triacylglycerols (TAGs) were also affected, but levels of maize diacylglycerols (DAGs) were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably involved at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since the expression of NPR1, POX12 were barely affected, PR4 after 6 h with BOA-4/7-OH and PR1 after 24 h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species, and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.For the characterization of BOA-OH insensitive plants, we studied the time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of The online version contains supplementary material available at 10.1007/s10886-021-01337-z. DIMBOA but also DIBOA -one) which is, for instance, present in rye and old maize leaves, possess biocidal properties, influence growth of plants and microorganisms, and can function as a toxin for sensitive herbivores -one is the first degradation product of DIBOA. Less sensitive plants can hydroxylate BOA in position 5 or, preferentially, in position 6, and immediate glucosylation yields BOA-5/6-O-glucoside as a minor or major detoxification product , which is highly resistant to BOA when cultured in soils rich in organic matter, accumulates low amounts of BOA detoxification products, compared to maize, although roots of A. theophrasti can detoxify BOA via BOA-6-OH glucosylation by a cell wall associated glucosyltransferase  containing Acanthus ilicifolius plants  which are stored in lipid droplets in the cytosol, play a role in stress responses  phosphatidylcholine (PC) as the dominant PL, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), the glycolipids (GLs) monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and low amounts of free fatty acids. Plasma membranes of maize roots are composed predominately of PC and PE, whereas PG, PI, and PS are less abundant  and catalase (CAT) gene expressions for ROS detoxification and on desaturase FAD2 genes for unsaturated fatty acid regeneration in the youngest and most vulnerable root tissue exhibiting responses on the lipid level were studied. We searched for a coordination of membrane repair, ROS elimination, BOA-OH detoxification, and presumable links to pathogenesis and resistance in maize root tips. The study aimed to obtain better insights in the BOA-OH responses of maize known to be insensitive to BOA-6-OH and whether BOA-OH isomers differ in the time-dependent effects on maize.In this study, we describe the synthesis of four BOA-OH isomers as compounds causing oxidative stress in maize root tips, and time-dependent differentiated reactions of the root zones faced with these compounds. We looked for early impacts of BOA-6-OH on root membrane lipids of maize in comparison to the weed Except for BOA-6-OH, hydroxylated benzoxazolinones are commercially not available. Therefore, the isomers of hydroxylated benzoxazolinones, including BOA-6-OH, were synthesized to compare their detoxification in maize seedlings and to study gene expression. However, the laborious syntheses and yields of the different isomers did not allow to perform all experiments with all isomers.1) was prepared by nitration of resorcinol with conc. H2SO4 and 67% HNO3. 1 was isolated by steam distillation as orange crystals . The melting points (mp) of 85 °C was in accordance with Schaffrath  δ 10.65 , 7.44 , 6.61 . 13C-NMR  δ 156.5 . 138.9 , 123.9 , 109.6 .2-Nitroresorcinol (haffrath . NMR spe2) was prepared by the classical Elbs oxidation of 2-nitrophenol with K2S2O8 in diluted NaOH solution  and a final crystallization of the MTBE extract from water. The mp of 131–132 °C is in accordance with the data of Elbs  δ10.14 , 9.60 , 7.22 ,7.02 , 6.96 . 13C-NMR  δ149.7 , 145.1 , 135.8 , 124.3 , 120.1 , 109.5 .2-Nitrohydroquinone (ion Elbs . 2 was o of Elbs . 1H-NMR 3) was prepared from resorcinol according to the recent nitration method of Samajdar et al. (3)3 × 5 H2O with some variations . 3 was isolated by extraction with CHCl3 and purified by flash chromatography over silica gel (eluent n-hexane/ethyl acetate 2:1 v/v) as yellow crystals in 26% yield and 2 g amount. The mp of 120 °C is in accordance with Kauffmann and Kugel  δ 10.95 , 10.80 , 7.88 , 6.42 , 6.39 . 13C-NMR  δ 164.9 , 156.0 , 128.1 , 127.8 , 108.8 , 103.6 .4-Nitroresorcinol  was prepared by nitration of an ethereal solution of catechol with 100% fuming nitric acid according to Rosenblatt et al.  δ10.62 , 7.65 , 7.24 , 6.91 , 5.82 . 13C-NMR  δ 146.5 , 142.8 , 133.8 , 121.7 , 119.8 , 115.8 .3-Nitrocatechol  were dissolved in 150 ml of dry THF followed by 0.5 g Pd/C catalyst. The mixture was stirred under hydrogen from a balloon at standard pressure for 6 h. The H2 balloon was then removed, the flask was cooled in an ice water bath and 2.3 g  triethylamine was added at once followed by dropwise addition of 0.8 g (2.6 mmol) triphosgene dissolved in 50 ml dry THF over a dropping funnel within 3 min. The mixture was stirred for 30 min. Then, the catalyst and the triethylamine hydrochloride were filtered. The solvent was removed from the filtrate in vacuo. The remaining residue was purified by column chromatography using CH2Cl2/methanol 10:1 as the eluent. An exception is compound 7. All melting points given here were in accordance with those reported by Zinner and Wigert . The IR-spectra were obtained with a Fourier Transform Infrared Spectrometer FT/IR-4100 (JASCO). The EI-MS data were acquired on a MAT 8230 spectrometer . Melting points were determined with a Boëtius micro hot stage.In a glass hydrogenation flask, 1.54 g (9.9 mmol) of the corresponding nitro compound -one 5: Yield: 998 mg (66%) off-white crystals, mp. 288–289 °C  δ10.69 , 6.86 , 6.71 , 6.63 . 13C-NMR  δ 154.5 , 144.7 , 142.0 , 122.0 , 118.2 , 110.9 , 100.9 . IR (KBr): 4-Hydroxybenzoxazolin-2(3H)-one 6: Yield: 651 mg (43%) beige crystals, mp. 212–213 °C  δ 11.33 , 9.36 , 7.02 , 6.47 , 6.41 . 13C-NMR  δ155.1 , 154.1 , 136.3 , 131.0 , 109.9 , 108.0 , 97.4 .IR (KBr): 5-Hydroxybenzoxazolin-2(3H)-one 7: A black solid remained after removal of the solvent in vacuo. It was refluxed for 10 min along with 0.5 g activated charcoal powder and 200 ml of distilled water. It was then filtered and cooled to yield 250 mg (17%) of 7 as colorless crystals, mp 299 °C  δ 11.24 , 9.36 , 6.86 , 6.69 , 6.55 . 13C-NMR  δ154.8 , 153.1 , 144.1 , 122.3 , 110.1 , 109.9 , 98.0 . IR (KBr): 6-Hydroxybenzoxazolin-2(3H)-one 8: Yield: 420 mg (29%) beige crystals, mp 236–238 °C  δ 10.50 , 6.90 , 6.56 , 6.51 . 13C-NMR  δ 154.5 , 140.9 , 131.8 , 131.1 , 124.2 , 110.3 , 110.9 .7-Hydroxybenzoxazolin-2(3IR (KBr): Zea mays cultivar Cassila  were hydroponically grown for 7 days as described in Schulz et al. .Caryopses of z et al. . A. theoO-glc accumulation during short-term exposure, seedlings were incubated 0, 10, 20, 30, 40, 50, and 60 min with 0.5 µM BOA-6-OH. Analysis and determination of the detoxification products were performed by HPLC using the system and the method described below. For preparation of 500 ml incubation solution, the isomers were pre-solved in 1 ml methanol and sonicated for 5 min before adding tap water. Incubations with 9 seedlings/60 ml were repeated at least 6 times for each BOA-OH isomer. Browning reactions were monitored after 30 min, 1 h, 6 h, and 24 h, bubble formation after 10 min, 30 min, 1 h, and 6 h. Root extracts were performed as described in Schulz and Wieland (1998). Peroxidase activity at living root surfaces was detected by bathing the roots  in 10 ml 0.1 mM acetate buffer pH 5.0 supplemented with 1.6 ml 2 mM ABTS  and 0.6 ml 15 mM H2O2 . Sites with peroxidase activity immediately turned dark or black-green. Catalase activity elicited by BOA-6,5,4-OH was concluded from bubble formation according to Schellhorn and Stones . For analysis of BOA-6-d Stones  and Wheed Stones , present2O and methanol within 35 min. For confirmation of the compounds identity, root hair extracts were subsequently analyzed by Ultra Performance Liquid Chromatography (UPLC)-electrospray (ESI)-mass spectrometry  as described in Schulz et al. . From another six seedlings, root hair zones were washed with 500 µl water, and the wash solutions including the mucilage drops (see below) collected in pure methanol, yielding a 50% solution. Four wash solutions collected over a period of 4 h were combined for one sample. Root hair extracts and wash solutions were analyzed by HPLC/ DAD , equipped with a RP-18 column . Linear gradients were run with 0.01% formic acid/Hz et al. . MucilagA. theophrasti were harvested without incubation and after 10, 20, 30, 40, 50 and 60 min incubations in 0.5 mM BOA-6-OH. For these experiments, BOA-6-OH was purchased from Merck-Sigma. The harvested root material from each incubation time was immediately treated with boiling water for 20 min, followed by 3 × repeated chloroform/methanol (1:2), (1 ml/sample) extractions. The resulting three portions of crude lipid extracts were combined and purified by the addition of 0.75 ml 0.3 M ammonium acetate. After centrifugation at 2000 g for 5 min, the lipid phase was dried under N2. When not directly analyzed, the extracts were stored at -20 °C until analysis. The extracted root material was dried overnight at 104 °C for dry weight determination. The described extraction method followed the procedure described by Siebers et al. .For total fatty acids methyl ester (FAME) determinations, 35 root tips  /sample and 10 older root parts /sample from maize and 13 root tips (AbRTa)/ sample and 7 older root parts (AbRTb) /sample from s et al. . The cons et al. . For quas et al. . The oveA. theophrasti were extracted as described. Lipid extraction and analysis were according to Bravo et al.  analyses, and determinations of TAGs and DAGs, 4 maize root parts/sample (see above) or 7 root parts/sample of o et al. . Lipids o et al. . The frao et al. . The conSOD2, CAT 1, CAT 3, FAD2-1, FAD2-2, PR1, PR4, and POX 12 because of the limited amounts of BOA-4, 5- and 7-OH available for the experiments. For the same reason, the time points for real-time PCR were restricted, but within the most reactive period during the incubation regarding early gene responses, starting polymerization, and bubble development. The set of genes was selected to give first clues on the oxidative stress elicitation by BOA-OH isomers, lipid repair, and possible relations to plant immune responses in the youngest root tissues, thought as a preliminary base for future in-depth investigations. Superoxide dismutase 2 (SOD2) involved in superoxide radical transfer to H2O2 was included as an indicator for ROS stress  gene is responsive to H2O2, reactive oxygen species, and xenobiotics. Transcripts were detected in the root, epicotyl, and leaves . Expression of members of the complex gene families with functions in the glutathione–ascorbate (GSH–ASC) cycle, such as ascorbate peroxidase producing H2O from H2O2, could not be addressed in this study. The gene expression of two ER-bound oleoyl desaturases (FAD2-1 and FAD2-2) was studied in context with lipid restoration processes after BOA-6-OH exposure. The enzymes synthesize linoleic acid (18:2) mostly in non-photosynthetic tissue  and immediately frozen in liquid nitrogen until RNA was extracted. The material was homogenized with the Precellys® 24 / 24-Dual  and RNA was isolated with the NucleoSpin RNA Plant Kit  according to the instructions of the manufacturer. For cDNA preparation, the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit was used. RT-PCR was performed with a 7300 real-time PCR system . The reaction mixture (20 µl) consisted of 4 µl H2O, 5 µl cDNA, 1 µl primer pair (working solution 10 pmol/µl) and 10 µl 5xEvaGreen (ROX)qPCR-Mix II . Gene expression was determined relative to the control plants and to ZmActin as reference gene. For evaluation of RT-qPCR results, the ΔΔct method was applied.7-day-old maize seedlings were incubated with 0.5 mM of the BOA-OH isomers for 30 min, 1 h, and 6 h, for Bars, presented with SD, were established with data obtained from three biological replicates unless otherwise noted. Statistical analysis of all the data was done with the t-test. Data in the figures are presented as the mean ± standard deviation. Significant differences between samples and the controls are indicated: ∗ p < 0.05; ∗  ∗ p < 0.005; ∗  ∗  ∗ p < 0.0005.H)-ones (5–8) were prepared from the corresponding nitro precursors 1–4 + 396 (6), 379 (4), 364 (3), 350 (4), 332 (3), 278 (4), 231 (48), 203 (25), 185 (100), 137 (8), 110 (33); DIMBOA: 212 (M + H)+, 234 (M + Na)+, 250 (M + K)+ 212 (5), 194 (8), 180 (9), 177 (34), 162 (32), 151 (36), 139 (18), 125 (8), 111 (100); MBOA: 164 (M-H)−, 166 (M + H)+, 188 (M + Na)+ 166 (100), 151 (45), 138 (8), 123 (10), 122 (25), 110 (63), 107 (17), 95 (5). BOA-6-OH or its glucoside was not detected in any of the samples. With this background, the chosen BOA-OH concentration of 500 µM for the subsequent experiments was thought to be high enough to elicit stress reactions in the root tip.Emerging lateral roots destroy epidermal tissue, which leads only to a transient release of benzoxazinoids , linoleic acid (18:2), and α-linolenic acid (18:3). Under stress, the accumulation of free fatty acids in plant membranes is increased. Since unsaturated fatty acids (UFAs) are particularly susceptible to oxidation, we started with the determination of total UFAs in comparison to saturated fatty acid contents in the youngest (RTa) and older root tip parts (RTb).In the maize RTa and RTb samples, linoleic acid (18:2) is the main UFA, followed by α-linolenic acid (18:3), which is similar in amount as found for oleic acid. The analyses of the ZmRTa samples disclosed a significantly decreased free linoleic acid content 20 and 30 min after BOA-6-OH exposure. Regeneration of the t0 level was already accomplished after 40 min and at 60 min, no further decrease was observed. Except for a significant increase of oleic acid after 40 min, there was no other effect on α-linolenic acid and oleic acid contents during the entire course of incubation. In the ZmRTb samples, linoleic acid increased in tendency during BOA-6-OH exposure, but the effect was not significant. In contrast, α-linolenic acid increased significantly after 10 min and stayed constantly higher during the next 40 min, then dropped to the t0 value. During this period, also the amounts of oleic acid was higher. In both series, significant changes in the contents of saturated fatty acids were not detected  of both UFAs after 20 and 30 min was found. Their regeneration to t0 levels was reached after 40 min. A significant decrease of UFAs was observed in the AbRTb samples after 20 and 30 min. The subsequent regeneration was as found for AbRTa  and stearic acid (18:0). Whereas stearic acid contents were not significantly affected, the ones of palmitic acid were reduced after 20 and 30 min of incubation with a regeneration to the t0 level after 40 min. The reduction was found in both sample series (AbRTa and AbRTb), with a stronger one in the youngest tissue, where a loss of about 50% of palmitic acid was measured. Thus, root tips of A. theophrasti are more affected than those of maize. Nevertheless, both species exhibit a remarkable fast regeneration of the fatty acid contents found at t0. The decrease of UFAs, and in case of A. theophrasti also the decrease of saturated fatty acids, is balanced already after 40 min.In RTa Fig. . Major sFor clues of structural phospholipid and glycolipid damage, their contents in the RTa/RTb series of both plants were determined and compared with the levels found in untreated roots set as t0 control Fig. . In extrIn the maize ZmRTa samples, only one of the major phospholipids, PC with the molecular species 34:2 and 34:4  and diacylglycerols (DAGs) contents were determined . In the AbRTb samples, similar fluctuation patterns were found, with increases after 50 min in AbRTb samples, compared to shorter incubation times. TAG 54.6 and 54.5 decreased during the first 20 min. For most of the molecular species, the t0 levels were not reached again after 1 h, in contrast to maize RTa samples. TAG levels in most of the t0 samples of the RTa series were often higher than the ones found during the incubation, which may indicate that TAG levels decreased already very early during the incubation. The up and downs in the TAG profiles may be related to different and cascaded organized regeneration sites.In A. theophrasti RTa samples, the main DAGs showed slightly reduced levels during the early course of the incubation, but recovered later. In the older root part, an increase of the levels between 10 to 50 min was found for the DAGs 16:0/18:3 and 16:0/18:2. In most of the A. theophrasti samples, DAG t0 levels were higher, however predominantly without significance. For both species, DAGs were less affected than TAGs. Since DAGs of A. theophrasti roots exhibited some responsiveness, PA values were here determined. PA amounts were only enhanced after 20 min in series a, and after 10 min in series b, otherwise not significantly affected  were found. In the ZmRTb sample series, DAGs, except for molecular species 18:2/18:2 at 40 min, were not significantly altered, although there was a tendency for reduction between 30–50 min. In the ted Fig. .Fig. 10DA. theophrasti and PC in maize are most affected with defined molecular species. 2) The highest response was ascertained for total fatty acids, particularly the decrease of UFA 18:2 in the youngest tissue in both species. UFA 18:3, not affected in ZmRTa samples, increased in ZmRTb samples, but considerably decreased in both A. theophrasti series. Palmitic acid was only reduced in A. theophrasti. 3) Regarding the neutral lipids, TAGs were affected in both species, but DAGs were almost not changed in maize, and A. theophrasti to a much lower degree than TAGs. 4) Disturbances of membrane PL contents were rapidly balanced in young root tissue of both species. The data indicate a) different PLs with defined molecular species as major targets for BOA-6-OH damage and b) differences in membrane repair of maize and A. theophrasti.The lipid analyses disclosed 1) an early decrease of defined membrane phospholipids, from which PI in SOD2 was upregulated in the root tip when the seedlings were exposed to the BOA-OHs, shown in Fig. SOD2 expression after 30 min of incubation. The high levels were maintained over a period of 6 h, then dropped to 4–4.5fold after 24 h. With BOA-7-OH, a retarded increase to a similar extent was found (6.5fold after 24 h).Gene expression studies were performed by qRT-PCR to unravel whether ROS caused oxidative stress and the changes in lipid composition were based on transcriptional regulation. The expression of FAD2-2 gene expression during BOA-6-OH exposure was increased after 30 min up to more than fourfold and maintained at higher levels for at least 6 h. The up regulation of FAD2-2 expression was higher with BOA-5-OH isomers and reached a sevenfold expression after 6 h  and of BOA-7-OH (3.2fold). Thus, the expression profiles of CAT3, PR4, and PR1 seem to be specific for the isomers, while the abundant naturally occurring BOA-6- and BOA-5-OH had similarities in their effects. The response may be specific for the concentration and presumably dependent on the duration of exposure.Maize seedlings started to glucosylate BOA-6-OH almost immediately when exposed to the compound. Within 10 min, about 100 nmol/g FW of the glucosides were produced in the root tips. The amounts of the detoxification product increased constantly further on, reaching about 500 nmol/g FW within 1 h.O-glucoside and ~ 3 µmol BOA-5-O-glucoside accumulated in the roots, but only traces of not further analyzed BOA-4-OH and BOA-7-OH detoxification products  and BOA-7-OH (6%), indicating that additional mechanisms contribute to the compounds´ detoxification or elimination by polymerization. The position of the OH group strongly influenced the detoxification via glucosylation.For the long-term accumulation of detoxification products, also the other isomers were tested. Within 24 h, ~ 5 µmol/g FW BOA-6-cts Fig. . The fasA. theophrasti developed bubble formation as a response to BOA-OH treatment which was, however, not restricted to root hairs, but covered the entire root during the early phase of incubation . Cruz-Ortega et al. , might be suppressed in maize but perhaps not in A. theophrasti to a similar degree. According to Liu et al. , . Only the most abundant naturally occurring BOA-6-OH and BOA-5-OH led to a low enhancement of PR4 expression after 6 h, indicating that linolenic acid derivatives could be too low to stimulate the JA defense signaling pathway efficiently. We believe that JA precursors from nonenzymatic peroxidation of linolenic acid do not accumulate in sufficient amounts to trigger plant defense reactions considerably. On the other hand, cleavage of linoleic acid (18:2) or linolenic acid (18:3) from membrane lipids can submit to pathways resulting in so-called death acids Below is the link to the electronic supplementary material.


X-ray powder diffractograms suggest the same structure also for X– = Br– and I–.In this work, we show that intramolecular hydrogen bonding can be used to stabilize tri-coordinated phosphane-gold(I) complexes. Two molecular structures of 2-(diphenylphosphino)benzoic acid (L) coordinated to a gold(I) atom were determined by single-crystal X-ray diffraction. The linear L–Au–Br shows a standard linear coordination and dimerizes via hydrogen bonds of the carboxylic acid. Upon addition of two additional phosphane ligands the complex [LThe online version contains supplementary material available at 10.1007/s00706-021-02843-2. In the latter case, the gold atom is often in a linear, sometimes more or less distorted environment with the halide non- or weakly bonded + or tetrahedral [(R3P)4Au]+ coordinated cation, respectively [3P)3AuX  [+ (L = phosphane or carbene ligand) and [(P^P)2Au]+ have been reported BF4 BF4 . However)4Au]BF4 . In thisanilide) . An exteanilide) . Similarnterions .2(o-BzOH)P, with equimolar amounts of (tht)AuCl (tht = tetrahydrothiophene) leads to the formation of 2-(diphenylphosphino)benzoic acid gold(I) chloride, Ph2(o-BzOH)PAuCl, 1-Cl. It should be mentioned that various gold(I) complexes bearing a diphenylphosphino-benzoic acid—both ortho and para—have been reported, among them also 1-Cl X , 2-X, is formed benzoic acid, Phlso 1-Cl –59. Thro2-X, a thorough characterization by NMR spectroscopy was only possible for 1-X. Nevertheless, we conducted 31P{1H} NMR studies to get some basic information by adding different equivalents of the phosphane ligand to solutions of 1-X in CDCl3 . The 31P NMR shift of the free ligand is at − 4.1 ppm. Upon adding (tht)AuCl in equimolar amounts, the peak is shifted to 36.1 ppm for 1-Cl. For 1-Br and 1-I the peaks can be found at 38.3 and 42.5 ppm, respectively. An additional equivalent of the phosphane ligand gives a very broad signals at ~ 45 ppm, the respective peaks of for the bromide (~ 42.5 ppm) and iodide (~ 39 ppm) are slightly sharper. These observations are indicative for a dynamic behaviour of the present gold(I) species, presumably due to an equilibrium between the neutral tri- and ionic di-coordinated form according to [AuL2X] ⇄ [AuL2]++ X– [L = PPh2(o-BzOH)]. After the addition of the third equivalent of the ligand, the 31P NMR signals either almost vanish  or have a low intensity  reflecting the low solubility of the complexes. Indeed, already with the addition of the second equivalent, some white precipitate forms. These observations are indicative for a lower solubility of the 2-Br/2-I compared to 2-Cl, which might be the reason for the unsuccessful attempts of growing single crystals of all prepared complexes (vide infra). Interestingly, upon addition of an excess of the ligand to 2-Cl, not only the peak of the complex, but also the free ligands are broad possible due to an exchange between bound and free phosphane ligands.Due to the low solubility of the tri-coordinate gold(I) complexes 1-Br, could be obtained by slow gas diffusion of n-pentane into a concentrated solution of 1-Br in DCM benzoic acid gold(I) bromide, of CHCl3 , hence 1omplexes , 63. Con present . The sho present . Fig. 2L2(o-BzOH))2I] could be isolated, however, we were not able to synthesis this compound in pure form. This is somewhat surprising as the homologue chlorido-complex is known X (L = 2-(diphenylphosphino)benzoic acid, X = Cl, Br, I). The molecular structure of [L3Au]Cl could be determined by single-crystal X-ray diffraction, whereas those of X = Br and I are deduced by a similar pattern of powder-diffractograms. We suggest intramolecular hydrogen bonds for facilitating tri-coordination of sometimes difficult to stabilize tri-coordinated gold(I) atoms. Further synthetic work has to prove the scope of this concept, particularly for complexes which are better soluble than 2-X. Formation of 2-X is clearly favoured by the low solubility of these complexes whereas in solution, both coordinated ligands as well as intramolecular hydrogen bonds would have to compete with possibly interfering solvent molecules.In this work, we suggest a new concept to stabilize coordination numbers larger than two of gold(I) in its phosphane complexes beyond the dominating standard linear coordination. Using intramolecular hydrogen bonds, it is possible to stabilize a complex of the form ), contain supplementary crystallographic data for this paper. This information can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/Single-crystal structure analysis was carried out at room temperature on a Bruker D8 Quest ECO diffractometer with graphite-monochromated MoKHELXS-97 ) and refL-2014/7 }. The H 3 DCM and 2-(diphenylphosphino)benzoic acid  was added and the mixture was stirred for 2 h at r.t.. Pentane was added until a white precipitate formed. Yield: 118 mg (71%); 1H NMR : δ = 8.31 , 7.66 , 7.51 , 6.96  ppm; 13C{1H} NMR : δ = 168.55 (s), 134.85 , 134.07 , 133.11 , 132.89 , 132.83 , 131.84 (s), 131.64 , 130.42 (s), 129.57 (s), 129.19  ppm; 31P{1H} : δ = 35.97 (s) ppm; HRMS(ESI): m/z for [M + Na]+ calculated 561.0056, found 561.0094. was prepared according to a modified literature method , 57:  was dissolved in 5 cm3 water. After combining both solutions, the reaction mixture was stirred for 2 h at r.t. Phases were separated and the organic phase was dried over anhydrous Na2SO4. Evaporation of the solvent resulted a white solid. Yield: 17.4 mg (91%); 1H NMR : δ = 8.30 , 7.56 , 7.51 , 6.96  ppm; 13C{1H} NMR : δ = 169.36 (s), 135.10 , 134.15 , 133.09 (s), 132.47 (s), 131.67 (s), 131.12 (s), 130.32 (s), 129.38  ppm; HRMS(ESI): m/z for [M + Na]+ calculated 604.9551, found 604.9593. Crystals suitable for single-crystal X-ray diffraction were obtained by slow gas-phase diffusion of pentane into a diluted DCM solution.  was dissolved in 5 cm3 DCM. KI  was dissolved in 5 cm3 water. After combining both solutions, the reaction mixture was stirred for 2 h at r.t. Phases were separated and the organic phase was dried over anhydrous Na2SO4. Evaporation of the solvent resulted in a white solid. Yield: 10.8 mg (91%); 1H NMR : δ = 8.29 , 7.65 , 7.52 , 6.96  ppm; 13C{1H} NMR : δ = 168.94 (s), 135.01 , 134.17 , 133.09 (s), 132.47 (s), 131.67 (s), 131.66 (s), 130.35 (s), 129.37  ppm; 31P{1H} NMR : δ = 42.5 (s) ppm; HRMS(ESI): m/z for [M + Na]+ calculated 652.9412, found 652.9468.  was dissolved in 5 cm2-Cl in chloroform.  was dissolved in dry DCM and cooled and 2-(diphenylphosphino)benzoic acid  was added to the solution and stirred for 2 h at r.t. The product directly precipitated from the reaction solution. Filtration of the product resulted in a white solid. The product was dried in vacuum. Yield: 217 mg (86%). Due to the low solubility of the compound, no NMR and MS spectra could be recorded. Crystals suitable for single-crystal XRD were obtained by addition of excess amounts of the respective ligand to a slurry of   was dissolved in chloroform and 2-(diphenylphosphino)benzoic acid  was added. After a few minutes of stirring at r.t. the product precipitated directly from the reaction solution. Excess solution was decanted and the white powder was washed with chloroform. The product was dried in vacuum. Yield: 191 mg (95%). Due to the low solubility of the compound, no NMR and MS spectra could be recorded.  was dissolved in chloroform and 2-(diphenylphosphino)benzoic acid  was added. After stirring for a few minutes, the product began to precipitate directly from the reaction solution. Excess solution was decanted and the white solid was washed with chloroform. The product was dried in vacuum. Yield: 175 mg (93%). Due to the low solubility of the compound, no NMR and MS spectra could be recorded.Supplementary file1 (DOCX 314 KB)Below is the link to the electronic supplementary material.