N-heterocyclic carbene (NHC) complex in aqueous solution and in a range of alcohols to generate phenol and aryl ethers, respectively. The optimized reactivity showcases the ability to make targeted C—O bonds, but also identifies conditions where water and alcohol activation could be limiting for C—C and C—heteroatom bond-forming reactions. This copper(II) complex shows strong reactivity toward arylboronic acid activation in aqueous medium at ambient temperature. The relationship between product formation and temperature and catalyst loading are described. Additionally, the effects of buffer, pH, base, and co-solvent are explored with respect to phenol and ether generation reactions. Characterization of the new copper(II) NCN-pincer complex by X-ray crystallography, HR-MS, cyclic voltammetry, FT-IR and UV-Vis spectral studies is reported.Arylboronic acids are commonly used in modern organic chemistry to form new C—C and C—heteroatom bonds. These activated organic synthons show reactivity with heteroatoms in a range of substrates under ambient oxidative conditions. This broad reactivity has limited their use in protic, renewable solvents like water, ethanol, and methanol. Here, we report our efforts to study and optimize the activation of arylboronic acids by a copper(II)  In thatalysis –6. Coppeatalysis . Since tatalysis –10. Howeatalysis . The firatalysis . Recentl]2[SbF6] ,14. Buil]2[SbF6]  and Sing]2[SbF6]  also repPhenylboronic acids are effective cross-coupling agents for the generation of C—C, C—N, and C—heteroatom bonds . SpecifiHere, we report the synthesis and characterization of a pincer copper(II)-NHC complex for aryl boronic acid activation toward generating C—O bonds. This copper(II) NHC complex shows strong σ donor and π-back bonding properties, which are promising attributes for development of catalysts for cross-coupling reactions in sustainable solvents ,22. This2.2.1.Picolyl chloride (TCI), benzimidazole, phenylboronic acid , sodium carbonate, trisodium phosphate dodecahydrate , Sodium dihydrogen phosphate monohydrate, potassium carbonate, disodium hydrogen phosphate heptahydrate , ammonium hexafluorophosphate, triethylamine, potassium hydroxide , copper(II) acetate  were used as received. Perchloric acid, tetrabutylammonium hexafluorophosphate, and phosphorus(V) oxide were obtained from GFS chemicals, Inc. , SACHEM, Inc. , and Sigma-Aldrich , respectively. All reagents used for this work were analytical grade and used as received. HPLC grade acetonitrile was obtained from Fisher Scientific . The water used was initially deionized using a reverse osmosis system and further polished to be ~17 MΩ-cm.2.2.4. This solution was filtered, and the DCM was removed under reduced pressure. The oily residue obtained was washed with THF (2 × 10 mL), giving dark brown solid (1). 1H NMR : δ = 11.85 , 8.51 , 7.90–7.85 , 7.84 , 7.76 , 7.55–7.53 , 7.26–7.25 , 6.01  ppm : δ = 152.4, 149.7, 144.1, 137.8, 131.7,127.1, 124.0, 123.8, 114.2, 52.7 ppm , benzimidazole , and sodium carbonate  was mixed in 25 mL of ethanol in a 50 mL round bottom flask and refluxed for 36 h . The sol 2H) ppm ; 13C NMR52.7 ppm .2.3.2O, followed by the addition of 5 equivalents of ammonium hexafluorophosphate  imidazol-3-ium chloride  was dissolved in a minimum amount of H.5 mmol) . A brown  ; 13C NMR52.7 ppm .2.4.2  in MeOH (3 mL) and stirred for 1 h at 50 °C. A blue precipitate of complex 3 was obtained which was isolated by filtration and washed with cold MeOH. This precipitate was dissolved in acetonitrile and MeOH and kept for slow evaporation. Blue-green crystals suitable for X-ray diffraction were obtained from this solution after approximately a week at room temperature. ESI-HRMS (m/z): observed 422.36 for [M—CH3OH2014PF6−]+; calcd 423.10 for [M—CH3OH—PF6−]+ (max/nm in CH3CN (εmax/M−1 cm−1): 555(165), 226(8440) and 259(9480).1,3-Bis(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-3-ium hexafluorophosphate  was mixed with Cu(OAc)H—PF6−]+ ; λmax/nm2.5.3 (0.01 mmol) in 5 mL H2O or buffered aqueous solution. The reaction mixture was stirred at 70 °C for 6 h to yield phenol as the product. The reaction mixture was extracted in ethyl acetate and the % phenol conversion was obtained by GC-MS. Similar procedure has been followed for the conversion of phenylboronic acid to anisole and other ethers based on the different solvent media used. The % conversion of anisole and ethers formed were determined by using GC-MS by direct injection of the product mixtures. All reported % conversions represent the average value measured from 3 separate trials. Error and error bars reported here are one standard deviation of the mean recorded from these replications.Reactions were carried out in a 25 mL round bottom flask charged with phenylboronic acid (0.1 mmol) and copper complex 2.6.1H chemical shifts are reported vs. TMS and are referenced to the residual solvent peaks. Mass spectra were obtained using a Bruker UHPLC microTOF-Q II High Resolution MS system operating in ESI ionization mode. UV/vis spectra were obtained using an OLIS modernized HP 8452 UV/vis spectrophotometer. CV data were acquired with a CHI 620A (CH Instruments) electrochemical analyzer. The complex 2 solution was sparged with UHP Ar for 20 min to remove dissolved O2 and gas flow was maintained over the solution during CV acquisition. A blank CV was acquired prior the CV of the complex. The acetonitrile solution was scanned between −1.8 to 1.5 V vs. Ag/Ag+ reference electrode from the open circuit potential OCP, while that of HClO4 was scanned between −0.5 to 0.5 V vs. Ag/AgCl (Sat’d KCl). The aqueous CV potential is reported against normal hydrogen electrode (NHE) using the formula below:IR spectra were recorded using a Thermo Scientific Nicolet 6700 FT-IR. NMR spectra were obtained using a Bruker AVANCE III 500 MHz spectrometer at room temperature. 24H26CuF6N5O3P. Crystal data and structure refinement details for complex 3 are shown in 3 contains the supplementary crystallographic data for this paper. The GC-MS was recorded in a Shimadzu QP-2010S GC-MS.The X-ray intensity data were measured at low temperature (T = 100 K), using a three-circle goniometer platform with a fixed Kappa angle at = 54.74 deg Bruker AXS D8 Venture, equipped with a Photon 100 CMOS active pixel sensor detector. Monochromatized copper X-ray radiation (λ = 1.54178 Å) was selected for the measurement. The structure was solved in a centrosymmetric monoclinic unit cell; space group: P 1 2(1)/n 1, with Z = 4 for the formula unit, C3.1) in a 68% yield. The chloride counterion was exchanged with PF6− forming compound 2.The synthesis of the tridentate copper-NHC complex was accomplished by initially synthesizing the tridentate NHC ligand in a two-step procedure as shown in 2 and Cu(OAc)2 were dissolved in MeOH and heated to 50 °C for 1 h. A dark blue precipitate of complex 3 was obtained with a 75% yield  metal center is hexacoordinated by the two nitrogen and one carbon of the tridentate ligand and three oxygen atoms of the methanol and acetate units. The two nitrogen atoms (Cu1—N3 = 2.066 Å and Cu1—N4 = 2.112 Å) and one carbon atom (Cu1—C1 = 1.935 Å) of the tridentate NHC ligand and one oxygen atom (Cu1—O1S = 1.975 Å) of the acetate moiety occupies the equatorial square planar position of the octahedron while the other oxygen atoms of the methanol (Cu1—O3S = 2.287 Å) and acetate (Cu1—O2S = 2.758 Å) moiety occupy the longer axial positions  /4 and Rout = (2.758 + 2.287 Å)/2) suggests that the complex is tetragonally distorted octahedral  and [(CN2)Cu(Cl)(SO(CD3)2)]X; the copper(II)−C(carbene) distances 1.915(3) and 1.932(2) Å, respectively, fall at the short end of those of known copper(II) NHC complexes (2+ (1.889(4) Å) is even much shorter, although the copper center is clearly six-coordinate -NHC reported by Singer , and is M−1cm−1) . InteresM−1cm−1) . We attrM−1cm−1) . This isomplexes  16]. Ow. Owmax =ordinate  40]..max = 552+ and acetate ions, making it easier to displace acetate with either water or a phenyl unit needed for a productive reaction.In terms of the phenol generation observed here, catalyst loading plays a key role in reactivity; catalyst loading is proportional to phenol generation. Generally, phenol generation occurs within the first hours of a reaction, where additional time does not dramatically influence product production. Phenol production is highly dependent on the reaction temperature, where increasing the temperature increases the % conversion considerably. The highest phenol production was observed at 70 °C or higher. It was also observed that the addition of base supports phenol generation. The pH of the reaction showed the best yields under basic conditions, which is consistent with observations regarding adding proton acceptors to the reaction medium. Further analysis showed that phenol generation was enhanced under both acidic and basic conditions, and at neutral pH only a trace amount of phenol is generated. Under acidic conditions, some aspect of our catalyst could potentially be protonated, increasing the lability of one of the copper ligands. At acidic pH values, which enhanced the phenol generation, it seems that protonation of the acetate ion could perhaps decrease the Coulombic attraction between the CuBasic conditions effectively increase the hydroxide concentration, which leads to higher phenol generation. This is likely due to the deprotonation of a copper(II)-bound water molecule, which increases the nucleophilicity of the Lewis acid/copper-activated water moiety, allowing it to better react with the activated phenyl unit. Complex 3 can also undergo transitions to give 4- and 5-coordinated species during the reaction in solution due to the hemilability of the pyridine coordination modes. A reaction mechanism for this process can be postulated  that fol5.Together, these catalytic observations give a clear indication of the conditions for generating phenol from the activation of arylboronic acids using the reported copper(II) NHC complex in water. This C—O bond forming reaction has been shown to have significant merit on its own ,41–46, bSupplemental Materials


Staphylococcus aureus and predominately consists of molecular species with ≥16-carbon acyl chains in the 1-position and anteiso 12(S)-methyltetradecaonate (a15) esterified at the 2-position. The analysis of the growth media for PG-derived products shows S. aureus releases essentially pure 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-1′-sn-glycerol (a15:0-LPG) derived from the hydrolysis of the 1-position of PG into the environment. The cellular lysophosphatidylglycerol (LPG) pool is dominated by a15-LPG but also consists of ≥16-LPG species arising from the removal of the 2-position. Mass tracing experiments confirmed a15-LPG was derived from isoleucine metabolism. A screen of candidate secreted lipase knockout strains pinpointed glycerol ester hydrolase (geh) as the gene required for generating extracellular a15-LPG, and complementation of a Δgeh strain with a Geh expression plasmid restored extracellular a15-LPG formation. Orlistat, a covalent inhibitor of Geh, also attenuated extracellular a15-LPG accumulation. Purified Geh hydrolyzed the 1-position acyl chain of PG and generated only a15-LPG from a S. aureus lipid mixture. The Geh product was 2-a15-LPG, which spontaneously isomerizes with time to a mixture of 1- and 2-a15-LPG. Docking PG in the Geh active site provides a structural rationale for the positional specificity of Geh. These data demonstrate a physiological role for Geh phospholipase A1 activity in S. aureus membrane phospholipid turnover.Phosphatidylglycerol (PG) is the major membrane phospholipid of IMPORTANCE Glycerol ester hydrolase, Geh, is an abundant secreted lipase whose expression is controlled by the accessory gene regulator (Agr) quorum-sensing signal transduction pathway. Geh is thought to have a role in virulence based on its ability to hydrolyze host lipids at the infection site to provide fatty acids for membrane biogenesis and substrates for oleate hydratase, and Geh inhibits immune cell activation by hydrolyzing lipoprotein glycerol esters. The discovery that Geh is the major contributor to the formation and release of a15-LPG reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane phosphatidylglycerol. The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated. Escherichia coli, as a substrate for phospholipid:apolipoprotein transacylase (Lnt) (sn-glycero-3-phosphoethanolamine (2-acyl-LPE) is transported into the cell interior by LplT , the most abundant membrane phospholipid in this organism. The composition of lysophosphatidylglycerols (LPG) and how they are metabolized or reintroduced into the biosynthetic pathway has not been studied in S. aureus.Phospholipids are the major building blocks of biological membranes. Their deacylated forms are called lysophospholipids, which perform a number of signaling functions  and partse (Lnt) , 6. Lnt  by LplT  and the  by LplT , 9. Stapte genes . S. aureS. aureus is an important human pathogen that secretes a wide spectrum of protein factors that promote virulence (SAUSA3300_0320) is a well-known lipase that is one of the most abundant exoproteins -methyltetradecanoyl-sn-glycero-3-phospho-sn-1′-glycerol (a15-LPG) that is released into the extracellular environment.irulence , 12. Glyproteins  that is isolates . The tracription to18, scription , 20, andcription . The prithe cell . The extthe cell , 23. Thein vitro  and bothin vitro . Becausein vitro –18, mostn vitro –. Here, wS. aureus PG has a strict positional distribution of fatty acids when grown in rich broth (–sn-2-position is dominated by 12(S)-methyltetradecanoic acid (a15), and the sn-1-position contains ≥16-carbon acyl chains (≥16) giving rise to the prototypical PG molecular species distribution annotated in Inset) reflects their relative abundance in the PG molecular species  grown in rich broth. The number above the line is the total carbon number of the PG molecular species and the 1-position/2-position acyl chains are indicated below the line. The PG molecular species have ≥C16 acyl chains at the 1-position and a15 in the 2-position (red). Download FIG S1, TIF file, 0.5 MB.Annotated PG molecular species composition of Copyright © 2023 Subramanian et al.2023Subramanian et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 60 min to pellet exosomes, and the pellet and supernatant fractions analyzed for LPG  iso 15-carbon acyl chains. These data show that a15:0 is the predominant acyl chain in extracellular LPG.Mass spectrometry alone cannot directly determine whether a 15-carbon LPG has a straight chain or has an tructure . The antfrom Val . The 15-from Val  and the from Val  predomin10.1128/msphere.00031-23.4FIG S213C6]isoleucine, [d3]leucine and valine. The major peak of 15:0-LPG was +5 (green) which would be derived from [13C6]isoleucine. The +3 15:0-LPG (red) would be derived from leucine. The unlabeled 15-LPG (black) was synthesized by the de novo pathway. Representative data from three independent experiments is shown. Download FIG S2, TIF file, 0.2 MB.Verification of a15-LPG structure. Strain AH1263 (wild-type) was grown in minimal media containing oleate and the PG fraction was isolated by solid-phase chromatography. Oleate is selectively incorporated into the 1-position of PG  as the substrate at pH 6.0 followed by immediate characterization of the products by LC-MS/MS. The 1- and 2-position LPGs are separated from each other in the liquid chromatography fractionation step in the LC-MS/MS workflow  38). Th. Th38). 39–39–detected . S. aurefugation . The isofugation  illustrafugation .10.1128/msphere.00031-23.6FIG S4S. aureus membrane PG. Membranes from S. aureus strain NE1360 (mprF::ϕNΣ) were isolated by lysostaphin treatment, hypotonic lysis and ultracentrifugation. The membrane fraction (180 μg protein) was then incubated at 37°C for 1 h with proGeh (60 μM) and the formation of a15-LPG determined by LC-MS/MS. Red trace, membranes alone; blue trace, with proGeh. Download FIG S4, TIF file, 0.10 MB.Geh hydrolysis of Copyright © 2023 Subramanian et al.2023Subramanian et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00031-23.7FIG S5FIG S5, TIF file, 0.1 MB.The pH dependence of positional isomerization in lysophospholipids. Reactions were carried out using Geh with 1-16:0-2-18:1-PG as the substrate for 15 min. The samples were either immediately injected into the LC-MS/MS system without concentration or workup, or left to stand for 12 at room temperature. (A) proGeh reaction at pH 8.5 followed by the immediate injection of the sample onto LC-MS/MS system. (B) proGeh reaction at pH 8.5 followed by the injection of the sample onto LC-MS/MS system after 12 h at room temperature. (C) proGeh reaction at pH 6.0 followed by the immediate injection of the sample onto LC-MS/MS system. (D) proGeh reaction at pH 6.0 followed by the injection of the sample onto LC-MS/MS system after 12 h at room temperature. Download Copyright © 2023 Subramanian et al.2023Subramanian et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. aureus reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane PG  for extracellular a15-LPG in ironment . These iironment , 49 suggironment –52. The holipids , is a coholipids , and stiholipids , 56. Valsn-2 chain of PG lies in the hydrophobic cavity occupied by the Orlistat β-side chain and the sn-1 acyl chain sits in the space occupied by the α-side chain of Orlistat (sn-1 ester is positioned adjacent to the active site serine with the carboxyl group sitting in the oxyanion hole formed by the backbone amide protons of Ser116 and Phe17 (sn-1 position of PG. The Geh homolog from S. hyicus (Shl) has robust lipase and phospholipase A1 activity  overlaid with the docked structure of PG (green). The sn-2 chain of PG lies in the cavity occupied by the Orlistat β-side chain and the sn-1 acyl chain sits in the space occupied by the Orlistat α-side chain. The PG head group (GroP) sits in the cavity occupied by the more polar amino ester side chain of Orlistat. S116 is the active site serine. (B) Comparison of the crystal structures of S. hyicus lipase/phospholipase A1  (grey) and S. aureus Geh (PDB ID: 6KSM) (orange). Ca2+  has a structural role and is located at the same position in both enzymes. The position of Orlistat (pink) in the Geh structure is shown. The only region that is slightly different in Shl and Geh is the lid domain  that opens and closes to allow substrate entry and exit from the active site. The R.M.S.D. difference between the Shl and Geh structures excluding the lid region are 1.101 Å and the full molecule R.M.S.D. is 1.562 Å. Download FIG S6, TIF file, 1.1 MB.Geh structure and PG binding. (A) Overlay of the structure of Orlistat (pink) bound to Geh , Bee venom PLA2 (P9297-Sigma), Orlistat (O4169-Sigma), [1-14C]oleate (specific activity 59 mCi/mmol) (Perkin Elmer).All chemicals and reagents were reagent grade or better. 17-LPG was added. For complementation experiments, strain AH1263 containing the vector pPJ480, strain PDJ171 (Δgeh) containing either the pPJ480 or pPJ630 vectors were grown in rich broth plus 10 μg/mL chloramphenicol to an A600 of 4.0. LPG was extracted from cells and media as described above. For heavy labeling of LPG, AH1263 cells were grown on defined media plates and grown overnight in defined media. The next morning, cells were washed with media containing no isoleucine, leucine or valine and then inoculated into defined media containing 0.5 μg/mL [13C6]isoleucine, 0.2 μg/mL valine and 0.5 μg/mL of [d3]leucine at an OD600 of 0.05, and then grown to an A600 of 1.0. LPG was extracted from cells and media as described above. AH1263 and PDJ171 cells were treated with 20 μg/mL of Orlistat and grown to a final A600 of 4.0. LPG was extracted from cells and media as described above.Strains AH1263, PDJ171 (Δgeh) , JE2, NE2; temperature, 500°C; collision gas, medium; ion source gas 1, 20 lb/in2; ion source gas 2, 35 lb/in2; decluttering potential, -80 V; and collision energy, -30 V. The multiple reaction monitoring transitions for LPG species are listed in LPG was analyzed using a Shimadzu Prominence UFLC attached to a QTrap 4500 equipped with a Turbo V ion source (Sciex). Samples were injected onto an Acquity UPLC HSS C18, 2.5 μm, 3.0 × 150 mm column at 30°C (Waters) using a flow rate of 0.2 mL/min. Solvent A was 5 mM ammonium acetate + 0.1% formic acid, and Solvent B was 95% methanol + 5 mM ammonium acetate + 0.1% formic acid. The HPLC program was the following: starting solvent mixture of 35% A/65% B, 0 to 1 min isocratic with 65% B; 1 to 3 min linear gradient to 100% B; 3 to 30 min isocratic with 100% B; 30 to 32 min linear gradient to 65% B; 32 to 35 min isocratic with 65% B. The QTrap 4500 was operated in the negative mode, and the ion source parameters were: ion spray voltage, -4500 V; curtain gas, 30 lb/in10.1128/msphere.00031-23.2TABLE S2Table S2, DOCX file, 0.01 MB.Mass transitions used to detect LPG molecular species by LC-MS/MS. Download Copyright © 2023 Subramanian et al.2023Subramanian et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 600 of 0.7 at 37°C with shaking at 200 rpm. The culture was cooled to 16°C and the cells were induced with 1 mM IPTG overnight with shaking at 200 rpm. Cells were harvested and resuspended in 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM imidazole. Cells were broken by two passages through a cell disruptor and centrifuged at 20,000 g for 45 min. Geh was purified from the supernatant using a Ni-NTA column by washing with 20 column volumes of each 20 mM Tris (pH 8.0), 200 mM NaCl containing 10 mM imidazole or 20 mM imidazole or 40 mM Imidazole. Geh was eluted with 20 mM Tris (pH 8.0), 200 mM NaCl, 250 mM imidazole. The eluant was further purified by gel filtration chromatography using a HiLoad 16/600 Superdex 200 column (Cytiva Life Sciences) equilibrated with 200 mM NaCl, 20 mM Tris, pH 7.5. The molecular weight was estimated by analyzing the peak size on a HiLoad 13/300 Super 200 analytical column (Cytiva Life Sciences). A standard curve was generated using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), RNase A (13.7 kDa), and aprotinin (6.5 kDa).pPJ628 (proGeh) and pPJ650 (mGeh) expression vectors were transformed into BL21(DE3) cells and grown in rich broth to an ODsn-glycero-3-phospho-(1′-rac-glycerol) by drying the lipid under N2 and hydrating with 50 mM Tris (pH 8.0) for 2 h at 37°C. Hydrated lipids were sonicated for 2 min twice in a water bath sonicator and then passed 30 times through an Avanti Mini Extruder with a 100 nm membrane. A 50 μL assay reaction mixture containing 100 mM bis-Tris (pH 6.0), 150 mM NaCl, 1 μM PG liposomes and 500 nM Geh was incubated at room temperature for 15 min and 50 μL of methanol containing 1% acetic acid was added to stop the reaction. The rection was centrifuged at 20,000 × g for 20 min and the samples were analyzed by LC-MS/MS. For PLA2 assay, a 50 μL assay reaction mixture containing 100 mM bis-Tris (pH 6.0), 50 mM CaCl2, 1 μM PG liposomes, and 0.5 μg PLA2 was incubated at room temperature for 1 h, and 50 μL of methanol containing 1% acetic acid was added to stop the reaction. The reaction was centrifuged at 20,000 × g for 20 min and the samples were analyzed by LC-MS/MS.In vitro activity of Geh and PLA2 was analyzed by mass spectrometry. Liposomes for Geh assays were prepared with 1-palmitoyl-2-oleoyl-14C]PG substate was isolated from strain NE1360 (mprf::ϕNΣ) labeled with 20 μM [1-14C]oleate (specific activity 59 mCi/mmol) for 2 h. Lipids were extracted using Bligh-Dyer method (2 and resuspended in 1 mL chloroform:methanol (1:1). [14C]PG was purified on a DSC-NH2 SPE column equilibrated with hexane. The column was washed with chloroform:isopropanol (2/1) and then with ether:2% acetic acid to elute fatty acids. [14C]PG was eluted with chloroform:methanol:0.8 M sodium acetate (60:30:4.5). The eluate was dried under nitrogen and extracted twice with chloroform: water (1:1). [14C]PG (1.5 × 106 dpm) was dried, hydrated with 50 mM Tris (pH 8.0) and liposomes were prepared as described above. Geh assays contained 50 mM Tris (pH 8.0), 150 mM NaCl, 35,000 dpm [14C]PG liposomes and 50 μM Geh in a final volume of 20 μL, and were incubated at 37°C for 30 min. A 20 μL PLA2 assay reaction mixture containing 50 mM Tris (pH 8.0), 1 mM CaCl2, 35,000 dpm [14C]PG liposomes and 0.4 μg PLA2 was incubated at room temperature for 30 min. A 15 μL aliquot of each reaction was spotted on a Silica gel H thin-layer chromatography plate developed with ethanol:chloroform:triethylamine:water (34:30:35:6.5); (vol/vol/vol/v) plus 1 mM EDTA. The thin-layer plate was exposed to a Phosphor screen and scanned by Typhoon PhosphoImager (Perkin Elmer). Membranes were prepared strain NE1360 by treating the cells with lysostaphin, followed by hypotonic lysis with sonication, and the membranes were collected by ultracentrifugation as described by Oku et al. . All solvent molecules and ligands except calcium and zinc ions were removed from the structure and Ser116 was mutated to Ala. After completion of docking processes, the obtained one of the poses was manually adjusted so that the carbonyl oxygen atom of cleavage site was located in the oxyanion hole. The fitted ligand and Geh (Ser116) structures were imported into MOE and prepared using QuickPrep and the ligand was minimized energy with carbonyl oxygen atom fixed in the oxyanion hole.The 1-palmitoyl-2-oleoyl-PG substrate was docked with Geh by using SwissDock server , 62. Forhttps://www.graphpad.com/scientific-software/prism/).Statistical tests were performed using GraphPad Prism software version 9.1.2 .All work with


N-substituted pyrrole derivatives has been presented by a reaction between 2,5-dimethoxytetrahydrofuran and various primary aromatic amines in the presence of NiFe2O4 anchored to modified carbon hollow microspheres (NiFe2O4@MCHMs) as a recoverable reactive catalyst. The Classon-Kass method has been used to synthesize the pyrroles in excellent yields and short reaction times in the same direction with green chemistry rules. This reaction was carried out by employing NiFe2O4@MCHMs as a catalyst to make a simple procedure with short activation energy in water as an accessible, non-toxic, and biodegradable solvent. This catalyst provides a promising pathway to synthesize N-substituted pyrroles several times in a row through the recyclability without remarkable loss of its catalytic activity. The NiFe2O4@MCHMs nanocatalyst was characterized by applying FT-IR, XRD, FE-SEM, TEM, EDS, BET, TGA, VSM, and elemental mapping techniques. Also, the synthesized N-substituted pyrrole derivatives were identified using melting point, FT-IR, and 1H NMR analyses.Pyrroles are widely spread worldwide because of their critical applications, especially pharmacology. An expedition method for one-pot synthesis of  It is predicted that more heterocyclic compounds containing nitrogen will be synthesized and used as drugs in the coming years.Nitrogen-containing heterocycles are played an essential role in biological compounds. Most of the drugs approved by the united states food and drug administration (FDA) and presently available on the market include nitrogen-containing heterocyclic moieties2. These compounds play a unique role in constructing natural structures and intermediates in many biological reactions3. Also, the pyrrole derivatives are current in diverse bioactive pharmaceutical molecules such as; anti-inflammatory4, antitumor agents5, anti-bacterial6, and immunosuppressants7. However, the applications of pyrroles are not limited to biological and medicinal compounds. Many derivatives of pyrroles, especially N-substituted pyrroles, are widely used in dyes8, agrochemicals9, polymerization10, and solar cells11.Pyrroles are a group of five-membered heterocycles that contain nitrogen in their structure. These heterocycles are among the most critical nitrogen-containing heterocycles in many natural products and drug compounds. Pyrrole-based materials are also finding increasing usage in materials scienceN-substituted pyrroles have been developed. N-substituted pyrroles can be obtained from a variety of raw materials. One of the most widely used raw materials for preparation of N-substituted pyrroles is 2,5-dimethoxy tetrahydrofuran. This method is known as the Klasson-Cass reaction12, can be prepared N-substituted pyrroles from the reaction between 2,5-dimethoxy tetrahydrofuran and amines. Banik et al., by using bismuth(III) nitrate as a catalyst synthesized the N-substituted pyrrole under ultrasonic conditions13. Zhang et al. synthesized a wide range of N-substituted pyrroles using an antimony magnetic catalyst in water as a solvent14. Naeimi et al. prepared the N-aryl pyrroles using ionic liquid, [H-NMP][HSO4] at room temperature in the green solvent15.Due to the widespread use of pyrroles, various synthetic methods for production of 19. They can reduce the reaction activation energy and time of the reactions21. One of the main factors that make new catalysts economically attractive is reduced reaction time and increased reaction efficiency25. Scientists are always looking for ways to improve catalysts29. Using nanoscience to design new catalysts revolutionized the use of high-performance catalysts31.The catalysts are essential in many chemical reactions32. One of the standard methods of classifying hollow structures is to organize them based on their shape and form33. Also, the hollow structures are classified based on their shell compositions. Hollow structures can exist in single-layer or multi-layer cylindrical, cubic and spherical forms. Hollow spherical structures can be categorized into monolayer spheres, multi-layer spheres34, and yolk shells35. Hollow spheres are widely used in drug delivery38, biosensors40, catalysts44, and batteries45. Many catalytic reactions have been carried out in hollow spherical structures46. Spherical hollow structures have a very high contact surface, which has led to their widespread use as a suitable substrate for the placement of nanoparticles. These NPs can be located on the inner and outer surfaces of spheres. This unique property significantly increases the active surface of the catalyst. Increasing the catalyst's active sites boosts the efficiency and reduces the reaction time.Hollow structures are a group of compounds named based on their texture and structure. Usually, a high percentage of hollow structures is the empty spaceN-substituted pyrroles. Furthermore, using the substrate, geometric structure and using the appropriate manufacturing method, and stabilizing metals on the surface of the substrate is one of the features of this project, which has made the designed catalyst have a unique recovery capability.The contact surface area of hollow spheres increases significantly when their walls are porous. Also, optimal use of the effective surfaces of the catalyst substrate increases the active catalytic sites and as a result, increases the reaction efficiency and significantly reduces the reaction time. The reaction between the raw materials on the surface, inside the channels inside the wall, as well as the inner surface of the hollow spheres, react together and produces the N-substituted pyrrole derivatives with the highest efficiency and minimum reaction times. Also, it was decided to recover the catalyst and reused several times. The reaction conditions are optimized in various parameters, such as; type of solvent, temperature, catalyst amount and the molar ratio of substrates.In this research, we hope to design, prepare and identify spherical hollow nanocatalyst for synthesis of −1. The Bruker DRX‐400 spectrometer recorded 1-hydrogen nuclear magnetic resonance (1H NMR) spectra in CDCl3 solvent and the tetramethylsilane as the internal reference. X-ray powder diffraction (XRD) patterns were reported by Philips X’Pert PW 3040 Powder X-ray diffractometer with CuKa radiation, and the patterns were analyzed with X’pert High score plus. The BELSORP-mini II apparatus  were calculated as nitrogen adsorption–desorption isotherms at 77 K, and the obtained data were measured by the Brunauer–Emmett–Teller (BET) method. The magnetic properties of the NiFe2O4@MCHMs catalyst were measured at room temperature using a VSM7300  in a maximum applied field of 15 kOe. The field emission scanning electron microscope (FE-SEM) of the surface, energy dispersive spectroscopy (EDS), and the map scan of modified carbon hollow microspheres were performed on TE-SCAN MIRA3 apparatus that operated at a 15 kV accelerating voltage.The reagents and solvents with high purity were purchased from Merck, Fluka, and Aldrich Chemical Companies. The used amine derivatives in chemical reactions were purified by the standard method. The melting points of synthesized organic compounds were determined with a Thermo scientific 9200-point apparatus. Fourier transform infrared (FT-IR) spectra were obtained as potassium bromide pellets with Thermo Nicolet IMPACT-400 FT-IR spectrophotometer in 400–4000 cm2 spheres are transferred to the freeze dryer at − 60 °C for 24 h. Finally, for carbonization, the dried carbon@SiO2 spheres were heated up to 450 °C for 6 h under pure argon atmosphere.At first, 60 mL of ethanol, 15 mL of deionized water, and 3 mL of aqueous ammonia (25%) were transferred to 250 mL round-bottom flasks equipped with a magnetic stirrer. After a homogeneous mixture was obtained, 2.88 mL of tetraethyl orthosilicate was added dropwise to the reaction mixture over 5 min. The reaction mixture was stirred at room temperature for 30 min with a magnetic stirrer. Then, to the reaction mixture added 1.1 mL of formaldehyde and 0.42 g of resorcinol. The reaction mixture was stirred at room temperature for 18 h. The brown precipitates were collected by the centrifuge at 5 min and 5500 rpm and washed three times with deionized water and ethanol. The wet carbon@SiO2 core of carbon@SiO2 spheres and prepare hollow carbon spheres, at first 60 mg of carbon@SiO2 spheres and hydrofluoric acid solution (20%) were transferred to PTFE (Teflon) beaker equipped with a magnetic stirrer. The mixture was stirred at room temperature for 6 h; finally, the precipitates were collected by centrifuge at 5 min and 5500 rpm, washed with deionized water and ethanol three times, and dried at 80 °C for 12 h.To remove the SiO. Then, it was transferred to a Teflon-lined stainless-steel autoclave for hydrothermal process, and placed in an electric oven at 200 °C for 2 h. The precipitates were collected by centrifuge at 5 min and 5500 rpm, washed three times with deionized water and ethanol, and dried at 80 °C for 12 h.First, 30 mg of hollow carbon spheres were dispersed in 100 mL of deionized water by ultrasound irradiation . Next, a mixture of 0.5 mmol of nickel (II) nitrate and 0.6 mmol of iron (III) nitrate was added to the hollow carbon spheres. After 5 min, 3 mL of aqueous ammonia (25%) was added to the mixture. The mixture was stirred overnight at room temperatureN-substituted pyrrole derivatives, a mixture of 2,5-dimethoxy tetrahydrofuran (1 mmol), aniline derivatives (1 mmol), NiFe2O4@MCHMs (2 mg), and distilled water (5 mL) were added to 25 mL round-bottom flasks equipped with a heater stirrer and stirred at 50 °C in oil bath. Periodic samples of the reaction were taken and analyzed using thin layer chromatography (TLC). After the reaction was completed and cooled to room temperature, the precipitates were dissolved in chloroform (3 mL). The catalyst was collected by an external magnet, washed with distilled water and acetone, and dried at 80 °C for reuse. A rotary evaporator removed the solvent to obtain the crude product. Finally, the crude product was recrystallized from ethanol to gain pure products.For the synthesis of 1H NMR analyses. Also, the synthesized compounds were named from 3a to 3l as follows.The synthesized organic compounds were characterized by melting point, FT-IR, and 1-phenyl-1H-pyrrole (3a); Brown solid; m.p.: 59–62 °C, decompose (Lit. m.p. 60–62 °C)47; IR (KBr): v = 3033 , 1497, 1597 (C=C Ar),746, 692  cm−1; 1H NMR  δ (ppm): 6.37 , 7.06 , 7.26–7.41 .1-(4-methoxyphenyl)-1H-pyrrole(3b); Black solid; m.p.: 87–89 °C, decompose (Lit. m.p. 88–89 °C)48; IR (KBr): v = 3033 , 2926 , 1597 , 1310 (C–O), 749 (C–H sp2 OOP) cm−1; 1H NMR  δ (ppm): 3.84 , 6.33 , 6.95 , 7.01 , 7.31.1-(3-methoxyphenyl)-1H-pyrrole (3c); Yellow solid; m.p.: 57–58 °C, decompose (Lit. m.p. 57–58 °C)49; IR (KBr): v = 3000 , 2850 (C–H sp3), 1400–1600 , 1300 (C–O), 743 (C–H sp2 OOP) cm−1; 1H NMR  δ (ppm): 3.86 , 6.36 , 6.81 , 6.95 , 7.01 , 7.10 , 7.31–7.35 .1-(4-bromophenyl)-1H-pyrrole (3d); Yellow solid; m.p.: 92–94 °C, decompose (Lit. m.p. 93–94 °C)50; IR (KBr): v = 3130 , 1497, 1590 , 727(C–H sp2 OOP), 512(C–Br) cm−1; 1H NMR  δ (ppm): 6.36 , 7.05 , 7.26 , 7.53 .1-(4-chlorophenyl)-1H-pyrrole (3e); Yellow solid; m.p.: 86–88 °C, decompose (Lit. m.p. 86.5–87.5 °C)50; IR (KBr): v = 3102 , 1501 , 728 (C–Cl) cm−1; 1H NMR  δ (ppm): 6.36 , 7.05 , 7.32 , 7.38 .1-(3-chlorophenyl)-1H-pyrrole(3f.); Black solid; m.p.: 51–52 °C, decompose (Lit. m.p. 50.5–51.5 °C)50; IR (KBr): v = 3102 , 1500 , 720–820 (C–Cl) cm−1; 1H NMR  δ (ppm): 6.36 , 7.07 , 7.21 , 7.27 , 7.33–7.37 , 7.40 .1-(2-chlorophenyl)-1H-pyrrole (3g); Black solid; m.p.: 85–86 °C, decompose (Lit. m.p. 86–87 °C)51; IR (KBr): v = 3050 , 1500 , 720–820 (C–Cl) cm−1; 1H NMR  δ (ppm): 6.43 , 7.18 , 7.61–7.65 , 7.74–7.76 , 8.11 .4-(1H-pyrrol-1-yl)phenol (3h); Brown solid; m.p.: 113–116 °C, decompose (Lit. m.p. 113–115 °C)52; IR (KBr): v = 3448 , 3135 , 1519 , 1024 , 732 (C–H sp2 OOP) cm−1; 1H NMR  δ (ppm): 6.18 , 6.51 , 6.80 , 7.16 , 7.31 .1-(p-tolyl)-1H-pyrrole (3i); Black solid; m.p.: 83–84 °C, decompose (Lit. m.p. 84–85 °C)53; IR (KBr): v = 3050 , 2922 , 1509, 1616  cm−1; 1H NMR  δ (ppm): 2.38 , 6.34 , 7.01 , 7.23 , 7.27 .1--1H-pyrrole (3j); Brown solid; m.p.: 119–120 °C, decompose (Lit. m.p. 120 °C)54; IR (KBr): v = 3054 , 1596, 1485 , 728 (C–H sp2 OOP) cm−1; 1H NMR  δ (ppm): 6.45 , 7.04 , 7.50–7.56 , 7.79 , 7.90–7.96 .1-(4-nitrophenyl)-1H-pyrrole (3k); Yellow solid; m.p.: 186–187 °C, decompose (Lit. m.p. 185–186 °C)53; IR (KBr): v = 3150 , 1616 , 1527 (N=O), 1347 (C–N) cm−1; 1H NMR  δ (ppm): 6.44 , 7.19 , 7.52 , 8.31 .1-(3-nitrophenyl)-1H-pyrrole, (3l); Yellow solid; m.p.: 75–77 °C, decompose (Lit. m.p. 75–76 °C)50; IR (KBr): v = 3150 , 1616 , 1527 (N=O), 1347 (CN) cm−1; 1H NMR  δ (ppm): 6.42–6.43 , 7.17–7.18 , 7.60–7.64 , 7.73 , 8.09 , 8.26–8.27 .2O4@MCHMs catalyst is consisted on the four main steps. In the first step, the SiO2 spheres were prepared as hard templates. Next, the SiO2 spheres were coated with a resorcinol–formaldehyde (RF) polymer to prepare carbon@SiO2 spheres. The wet carbon@SiO2 spheres were dried by the freezing method to preserve the porous structure of the resorcinol–formaldehyde (RF) polymer. The carbon@SiO2 spheres dried and then were heated to 450 °C under pure argon atmosphere to allow the porous channels on the surface of the spheres to enlarge.According to Fig. 2 inside the carbon@SiO2 spheres was removed by hydrofluoric acid (HF) to create a hollow structure. Finally, the prepared hollow carbon spheres were functionalized by NiFe2O4 using the hydrothermal method to enhance the surface and create the effective catalytic capability.In the next step, the SiOAfter preparation of the catalyst, various techniques including Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), field emission scanning electron microscopes (FE-SEM), energy dispersive X-Ray analysis (EDX), elemental mapping, Brunauer–Emmett–Teller (BET) theory, vibrating sample magnetometry (VSM), and thermogravimetric analysis (TGA) were used to study and identify the structure, morphology, and functional groups.2 and carbon@SiO2 spheres after being heated to 450 °C under pure argon, hollow carbon spheres, and NiFe2O4@MCHMs catalyst are shown in Fig. The FT-IR spectra of the carbon@SiO2 spheres  polymer chains. The peak was observed at 2902 cm−1 due to the stretching vibration of C–H sp3 bonding of the methylene group on RF polymer chains. Finally, the sharp peak that observed at 1104 cm−1 corresponds to stretching vibration of the Si–O bonding55.In FT-IR spectra of the carbon@SiO2 spheres after heated to 450 °C under pure argon .The diffraction pattern of the carbon@SiOres Fig. a shows ares Fig. a and cargon Fig. b, it canern Fig. c. Finall2O4@MCHMs catalyst's spherical shape is confirmed by the FE-SEM images  were 9.7241 nm and 0.7591 cm2O4@MCHMs catalyst were investigated using vibrational sample magnetometry  was used to study the thermal stability of the NiFeN-substituted pyrrole derivatives, the reaction between aniline and 2,5-dimethoxy tetrahydrofuran was chosen as a model reaction. The parameters such as; type of solvent, temperature, amount of catalyst, and the molar ratio between aniline and 2,5-dimethoxy tetrahydrofuran were investigated, and the related results are shown in Table To obtain the optimal reaction conditions and use them in the synthesis of 2O4@MCHMs catalyst as optimal conditions for the reaction model are selected. Also, the reaction efficiency decreased as catalyst amounts increased, which can be attributed to increased connections between the catalyst and raw materials.According to the results of Table 3O4@MCHMs, Ni@MCHMs, and NiFe2O4@MCHMs were prepared. Then, the reaction between aniline and 2,5-dimethoxy tetrahydrofuran was chosen as a model reaction and the product yield was calculated in the presence of each of the prepared catalysts and the related results are shown in Fig. 2O4@MCHMs demonstrate superior efficacy in comparison to those containing Fe3O4@MCHMs. Finally, the bimetallic sample has shown a much higher efficiency by taking advantage of the synergistic effect.To investigate the synergistic effect of the catalyst and its confirmation, the hollow carbon spheres, FeThe reaction was performed in the presence of aniline derivatives containing electron-donating and electron-withdrawing groups after optimizing the solvent, temperature, and catalyst amount.2O4@MCHMs catalyst. All of the N-substituted pyrrole derivatives prepared from aniline derivatives with electron-donating and electron-withdrawing groups had high yields and short reaction times. According to the Table Table 2O4@MCHMs catalyst was investigated with the previously reported catalysts for the synthesis of 1-phenyl-1H-pyrrole (3a) from 2,5-dimethoxy tetrahydrofuran and aniline, and the results are shown in Table 2O4@MCHMs catalyst compared to the reported catalysts. In comparison with of NiFe2O4@MCHMs catalyst, the investigated reported catalysts have lower efficiency in thermal conditions and the required more time to perform the reaction  was carried out on the catalyst both prior to and subsequent to its recovery. According to the findings, the NiFe2O4@MCHMs catalyst. An external magnet was used to separate the nanocatalyst from the reaction mixture after 2 min. The reaction progress was monitored using thin layer chromatography (TLC) after heating the filtrate mixture. The monitored reaction did not progress after filtration, so the hot filtration analysis shows the leaching for NiFe2O4@MCHMs catalyst does not happen.Furthermore, the hot filtration method was used to study the leaching of the NiFeN-substituted pyrrole derivatives has been investigated using new hollow nanocatalysts. Among the various methods for preparing N-substituted pyrroles, the article's authors have investigated one of the most efficient methods, i.e., the synthesis of N-substituted pyrrole derivatives from the reaction of the 2,5-dimethoxy tetrahydrofuran with various aniline derivatives. The prepared catalyst increased the reaction yield and also decreased the reaction times. In addition, the catalyst is easily separated from the reaction mixture and has displayed the ability to be reused with a minor decrease in efficiency. This hollow catalyst and its unique structure has increased the effective contact surface, which makes a smaller amount of catalyst needed for the reaction to progress. All the obtained products were identified using 1H NMR and have the highest degree of purity (Supplementary Information In this research, the synthesis of Supplementary Figures.


The binding of UPy-LL on the supramolecular nanoparticle system increased its antimicrobial activity against E. coli (MIC 3.53 µM to 1.77 µM) and improved its cytocompatible dosage for HK-2 cells from 5.40 µM to 10.6 µM. The system showed higher cytotoxicity (5.4 µM) to the macrophages. The high drug loading, efficient binding, enhanced antimicrobial behavior, and reduced cytotoxicity makes ION@UPy-NH2 an interesting drug carrier for AMPs. The combination with superparamagnetic IONs allows potential magnetically controlled drug delivery and reduced drug amount of the system to address intracellular infections or improve cancer treatment.Antimicrobial peptides (AMPs) can kill bacteria by disrupting their cytoplasmic membrane, which reduces the tendency of antibacterial resistance compared to conventional antibiotics. Their possible toxicity to human cells, however, limits their applicability. The combination of magnetically controlled drug delivery and supramolecular engineering can help to reduce the dosage of AMPs, control the delivery, and improve their cytocompatibility. Lasioglossin III (LL) is a natural AMP form bee venom that is highly antimicrobial. Here, superparamagnetic iron oxide nanoparticles (IONs) with a supramolecular ureido-pyrimidinone (UPy) coating were investigated as a drug carrier for LL for a controlled delivery to a specific target. Binding to IONs can improve the antimicrobial activity of the peptide. Different transmission electron microscopy (TEM) techniques showed that the particles have a crystalline iron oxide core with a UPy shell and UPy fibers. Cytocompatibility and internalization experiments were carried out with two different cell types, phagocytic and nonphagocytic cells. The drug carrier system showed good cytocompatibility (>70%) with human kidney cells (HK-2) and concentration-dependent toxicity to macrophagic cells (THP-1). The particles were internalized by both cell types, giving them the potential for effective delivery of AMPs into mammalian cells. By self-assembly, the UPy-coated nanoparticles can bind UPy-functionalized LL (UPy-LL) highly efficiently (99%), leading to a drug loading of 0.68 g g Inside the body, the particles meet phagocytic cells and nonphagocytic cells; therefore, the effect of the particles’ interactions on HK-2- and THP-1-derived macrophages was investigated by resazurin assay and, additionally, live/dead staining (−1 and 0.50 g L−1) showed cell viability greater than 70%, a threshold value for cytocompatibility  at high concentrations. The particles were internalized in both cells types. Because of these aspects, ION@UPy-NH2 represents a promising new drug carrier system.The cell experiments demonstrated good cytocompatibility (>70%) of ION@UPy-NHThe supramolecular interaction caused by hydrogen bonding of UPy units allows the addition of UPy-based drug molecules and generates an innovative magnetically controlled drug delivery system.2 was analyzed in binding experiments. UPy-LL can self-assemble with UPy-NH2 [−1) was bound. Compared to the binding of bare IONs with LL, where a maximal loading of 0.23 g g−1 LL on bare IONs with a starting concentration of 2 g L−1 was reached (efficiency: 11.5%), the supramolecular engineering highly improved the binding efficiency (99%) [2 (2-UPy-LL fibers. Cryo-TEM imaging of ION@UPy-NH2@UPy-LL showed particle agglomerates with directly bound short fibers with a size of 135 ± 55 nm (2@UPy-LL in which the particle concentration during adsorption was 0.4 g L−1.The binding of UPy-LL on ION@UPy-NH UPy-NH2 . After mcy (99%) . It is h(99%) [2 . Nile re(99%) [2 b. The sh ± 55 nm c. The foThe high binding efficiency allows the generation of a drug delivery system with high drug loading. Antimicrobial tests can be used to analyze if the drug is still active while being bound to the drug carrier.2, ION@UPy-NH2@UPy-LL, and free UPy-LL were analyzed by culturing these materials with a green fluorescent protein (GFP)-expressing E. coli . Therefore, the fluorescent bacteria were counted after 24 h. ION@UPy-NH2 without AMP led to comparable growth of the negative control (cells that were not combined with particles or AMP) for concentrations ≤ 0.03 g L−1 . Measurements of the growth with OD600 confirm these data  [2@UPy-LL.The antimicrobial properties of ION@UPy-NH03 g L−1 c. Higheriability . The str E. coli ,17,19. Iese data . Becausedsorbed) ,17. Beca2, the cytocompatibility of ION@UPy-NH2@UPy-LL and free UPy-LL was investigated (2@UPy-LL led to good cytocompatibility (>70%) until 10.6 µM (0.12 g L−1 particle concentration) for HK-2 cells and 3.53 µM for THP-1-derived macrophages (0.04 g L−1 particle concentration). At the respective particle concentration, no cytotoxic effect was measured for ION@UPy-NH2 , indicating the formation of hydrophobic pockets [We showed the detailed three-step synthesis route and characterization  of ION@UPy-NHous work . The bar work [2 . ION@UPy pockets .2 for 30 s at 65 W to induce a hydrophilic surface on the grid. The TEM grid was suspended using reverse-action tweezers, and using a micropipette, a 7 µL aliquot of 100× diluted sample was deposited on the grid and left under cover until the droplet was evaporated. Furthermore, the grid was kept under a high vacuum overnight, ensuring complete evaporation and minimizing imaging artifacts. For the DPC measurement the detector is segmented into different areas (A–D). The sample can deflect the electronic beam. Such deflection can create images where the A-C (DPCx (A–C)) and B-D (DPCy (B–D)) detector segments are differentiated. Cryo-TEM imaging was performed on samples with 0.5 g L−1 particles and incubated overnight in Millipore water using quantifoil carbon-covered grids . Before sample addition, grids were surface-plasma-treated (at 5 mA for 40 s) using a Cressington 208 carbon coater. Using an automated vitrification robot  operating at 22 °C and a relative humidity of 100%, a 3 µL sample was applied to the grids. The excess sample was removed using blotting filter paper for 3 s at −3 mm. The thin film formed was vitrified by plunging the grid into liquid ethane and subsequently, liquid nitrogen. The vitrified grid was transferred to a cryo-transfer holder and prepared for TEM imaging. TEM imaging was conducted using a CryoTITAN equipped with a field emission gun operating at an acceleration voltage of 300 kV, a postcolumn Gatan energy filter, and a 2048 × 2048 Gatan CCD camera. Vitrified films were observed with the CryoTITAN microscope at temperatures below −170 °C. Micrographs were taken at low-dose conditions using a defocus setting of −5 µm or −1 µm at 25 k magnification.High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and integrated differential contrast (iDPC) STEM imaging were conducted on a Thermo Fisher Scientific Titan Themis Cubed operating at an acceleration voltage of 300 kV and tuned with a monochromator and probe corrector. HAADF-STEM and iDPC imaging were acquired using Velox  software. For sample preparation, a Lacey carbon 200 mesh copper grid  was plasma-treated using a Gatan Solarus 950 Advanced plasma system  with O2 atmosphere with DMEM and RPMI medium, respectively. The differentiation of THP-1 monocytes into macrophages was induced by adding 50 ng/mL phorbol 12-myristate 13-acetate (PMA) to the culture medium and incubated for 48 h. The experiments were carried out with a seeding density of 2.5 × 105 cells/cm2 for the THP-1 cells and 2.5 × 104 cells/cm2 for the HK-2 cells.For cell culture, Dulbecco’s Modified Eagle Medium (DMEM (1×), gibco, ref. 42430-025) and Roswell Park Memorial Institute 1640 (RPMI (1×), gibco, ref. A10491-01) were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S), respectively. Human kidney cells  and monocytic human THP-1 cells  were cultured at 37 °C in 95% air/5% CO2, ION@UPy-NH2@UPy-LL (preparation can be found in 2.6 UPy-LL binding), and free UPy-LL for THP-1 macrophages and HK-2 cells was investigated by resazurin assay. The cells were seeded onto a 96-well plate (n = 3). The THP-1 cells were induced with PMA for 48 h, and the HK-2 cells were cultured overnight to allow cell adhesion. The particles were suspended in 70% EtOH by magnetic decantation and sterilized for two hours under UV light. Afterward, the supernatant was changed to PBS by magnetic decantation, removing the EtOH supernatant and resuspending the nanoparticles in PBS. The IONs were then ultrasonicated for 30 min in an ultrasonic bath to ensure full resuspension. The respective medium exchanged the buffer. After attachment, the culture medium was exchanged for a medium containing BIONs, ION@PGA, and ION@UPy-NH2 at the concentrations of 0 g L−1, 0.03 g L−1, 0.05 g L−1, 0.08 g L−1, 0.10 g L−1, 0.30 g L−1, and 0.50 g L−1, respectively, or ION@UPy-NH2@UPy-LL and UPy-LL at 0 µM, 1.77 µM, 3.53 µM, 5.40 µM, 10.6 µM, 14.1 µM, 17.7 µM, 26.5 µM, or 35.3 µM  at λex = 550 nm and λem = 584 nm. Results were presented normalized to the fluorescence of the negative control (cells that were not in contact with the nanoparticles or the AMP).The cytocompatibility of BIONs, ION@PGA, ION@UPy-NH2, ION@UPy-NH2@UPy-LL, and free UPy-LL was determined by live/dead staining. An amount of 50 µL of cell suspension was seeded (with a seeding density of 2.5 × 105 cells/cm2 for the THP-1 cells and 2.5 × 104 cells/cm2 for the HK-2 cells) into a 15-well Ibidi slide (n = 3). The particles were sterilized as described above in −1 or 0.50 g L−1 BIONs, ION@PGA, and ION@UPy-NH2 or 5.40 µM, 10.6 µM, or 14.1 µM ION@UPy-NH2@UPy-LL or UPy-LL. The cells were incubated for 24 h and washed with PBS (2×), incubated with 10 µM calcein AM and 10 µM propidium iodide-enriched medium , and washed with PBS (2×). Imaging acquisition of the cells was performed using a Zeiss Axio Observer 7 with a 10× objective.The cell viability of both cell types in combination with BIONs, ION@PGA, ION@UPy-NH2 containing UPy-Cy5 (−1 UPy-Cy5 solution in chloroform to 100 µL of an 11.6 g L−1 ION@UPy-NH2 suspension in water. The particles were shaken at 30 °C and 1500 rpm for 6 h and washed by magnetic decantation (3×) to ensure the binding. They were sterilized as described before and diluted to a 0.1 g L−1 suspension in the respective medium. An amount of 50 µL of THP-1 or HK-2 cells was seeded onto a 15-well Ibidi slide (n = 3). After incubation with ION@UPy-NH2/UPy-Cy5 for 5 min, 2 h, 24 h, and 48 h, the cells were washed with PBS (2×), fixed with 15 µL of 10% formaldehyde solution at room temperature (25 °C), and washed with PBS (2×). The cells were sequentially stained with Phalloidin Alexa 488 staining solution  for the membrane and 4′,6-Diamidin-2-phenylindol solution  for the nuclei in PBS, respectively. After washing (2× PBS), imaging was performed with a Zeiss Axio Observer 7 with a 40× objective.The internalization experiments of ION@UPy-NH UPy-Cy5  were pre2 with UPy-LL, 1.0 mg of UPy-LL was dissolved in 176.5 µL 99:1 MeOH:1× PBS pH 7.4 (gibco) containing 2 mM 2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) [−1 ION@NH2 solution in PBS. The particles were shaken at 1000 rpm overnight. The particles were sterilized under UV light for 30 min, and the buffer was changed by magnetic decantation.To combine ION@UPy-NH (HEPES) . An amou2 concentrations  in PBS were mixed 1:1 with various UPy-LL solutions . The experiment was conducted in triplicate. The suspensions were mixed for one hour at room temperature at 1000 rpm. After magnetic decantation, the supernatants were photometrically measured at 280 nm, and the concentrations of unbound UPy-LL were calculated with the help of a calibration line.To analyze the binding behavior, different ION@UPy-NH2 (bound to the IONs) and UPy-LL assemblies was examined by the Nile red (NR) encapsulation test on a Varian Cary Eclipse fluorescence spectrometer . A 0.1 g L−1 ION@UPy-NH2 suspension was combined with 0 µL, 0.08 µL, 0.16 µL, 0.32 µL, 0.64 µL, 1.28 µL, and 2.56 µL of UPy-LL as described above. After overnight incubation to let the fibers assemble, 0.53 µL of a 1 mM Nile red solution in MeOH was added (NR to UPy-LL 10:1). The particles were shaken at 400 rpm at room temperature for 20 min and then measured five times using a quartz cuvette .The formation of the UPy-NH2, ION@UPy-NH2@UPy-LL, and free UPy-LL was analyzed with (RH)4-GFP-expressing E. coli  performed in a manner analogous to the method stated in Turrina et al. [2 in 50 mM PBS at pH 7.4 was diluted to 0, 0.1 g L−1, 0.3 g L−1, 0.5 g L−1, 0.8 g L−1, 1.0 g L−1, 2.0 g L−1, 3.0 g L−1, 4.0 g L−1, and 5.0 g L−1. After dissolving, probes containing 0.0 µM, 2.2 µM, 4.4 µM, 8.9 µM, 18 µM, 35 µM, 54 µM, and 106 µM of the free UPy-LL were prepared. After binding, ION@UPy-NH2@UPy-LL was diluted to similar UPy-LL concentrations . During the experiment, all samples were finally diluted 1:10.The antimicrobial activity of ION@UPy-NHa et al. . ION@UPy2 is an interesting drug carrier material combing the favorable properties of IONs and supramolecular self-assembling systems. The UPy network forms a shell around the iron oxide core and small agglomerates, and it forms fibers. ION@UPy-NH2 and its precursor showed high cytocompatibility in HK-2 cells and a concentration-dependent effect on the metabolic activity of macrophagic THP-1 cells. The particles were internalized faster by THP-1-derived macrophages and were fully internalized after 24 h by both cell types. ION@UPy-NH2 binds UPy-LL with high efficiency (99%), inducing the reassembly and formation of smaller fibers. Attached to the drug carrier, the antimicrobial activity of UPy-LL is improved, leading to a MIC of 1.77 µM in E. coli. The combined system improved the cytocompatibility from 5.4 µM to 10.6 µM of the antimicrobial peptide for HK-2 cells, while it reduced the metabolic activity of THP-1-derived macrophages. The experiments demonstrated that ION@UPy-NH2 can be easily combined with UPy–drug molecules and could be used as a magnetically controlled drug delivery system for the antimicrobial peptide LL. The usage of low concentrations and improved cytocompatibility makes the AMP applicable.ION@UPy-NH


P. nigrum has been widely studied, research related to its possible immunomodulatory effects is relatively scarce. Here, the antitumor and immunomodulatory activity of an ethanolic extract of P. nigrum were evaluated in the murine models of 4T1 breast cancer and B16-F10 melanoma. In vitro evaluations showed that the P. nigrum extract has cytotoxic activity, induces apoptotic cell death, and has a pro-oxidant effect in both cell lines, but it regulates glucose uptake differently in both lines, decreasing it in 4T1 but not in B16-F10. P. nigrum extract significantly reduced tumor size in both models and decreased the occurrence of macrometastases in 4T1 model. Evaluation of immune subpopulations by flow cytometry revealed that the P. nigrum extract significantly increases the frequency of dendritic cells and activated CD8+ T cells and decreases the frequency of myeloid-derived suppressor like cells and Tregs in the tumor microenvironment of both models but with different dynamics. Our findings strongly suggest that the P. nigrum extract exerts immunomodulatory functions, slightly related to the modulation of cellular energy metabolism, which could ultimately contribute to the promising antitumor effect of P. nigrum.Although the antitumor effect of The online version contains supplementary material available at 10.1007/s00262-023-03487-3. Piper species, widely distributed in tropical and subtropical regions of the world, are used in the Ayurvedic system of medicine in India as well as in folk medicine in Latin America and the West Indies. Some species, like P. aduncum and P. hispidum are used for stomach pain and insect repellants. P. amalago distributed from Mexico to Brazil is used to relieve chest pain and as an anti-inflammatory agent [P. chaba and P. futokadsura are useful in asthma, bronchitis, fever, and hemorrhoidal conditions [ry agent , 2. The nditions .P. nigrum is widely used in folk medicine and has been used for diarrhea, indigestion, and respiratory system problems [P. nigrum has also shown significant in vitro and in vivo anticancer activity in many cancer models, including breast, prostate, colon, rectal, lung, melanoma, ovarian, and cervical cancers, among others [P. nigrum extract are alkaloids, including piperine, chavicine, piperidine, pellitorine, and piperedine, while flavones, flavanones, terpenes, steroids, lignans, neolignans, and alkamides are other main compounds [problems . Surprisg others . The majompounds , 7. Inteompounds .The ethanolic extract as well as the one obtained by supercritical fluids extraction, rich in piperines, present interesting cytostatic activity by inducing arrest in the G1 or G2/M phases of the cell cycle, respectively, in addition to differential cytotoxicity depending on the cell line, apparently due to a p53-dependent effect. Interestingly, the complete extract is more cytotoxic than the two main alkaloids, piperine and pellitorine, and has better specificity for tumor cells than the isolated compounds . A recenP. nigrum were evaluated in the metastatic 4T1 breast cancer and B16-F10 melanoma murine models. We found a significant reduction in tumor size in both models and a decrease in macrometastasis in the 4T1 model, along with an increase in the frequency of dendritic cells and activated CD8+ T cells. A reduced frequency of myeloid-derived suppressor-like cells (MDSC-LC) and Tregs in the tumor microenvironment was also evidenced. These results suggest that the modulation of the suppressive immune response could underlie the promising antitumor effect of P. nigrum extract.A recent review summarizes a large number of biological activities studied for piperines in cancer  and the Piper nigrum were collected in Putumayo, Colombia  and identified by Néstor García in the herbarium of the Pontificia Universidad Javeriana (voucher specimen number 30548). The plant material (80 g) was extracted as previously described [Fresh fruits of escribed . Brieflyescribed –16, was escribed , 18. Form/z range from 100 to 1800, using a full scan (Full-MS) and MS/MS. After the acquisition of the LCMS chromatograms, the raw data were exported in.mzXML format and processed in the free software MZmine version 2.53 (http://mzmine.github.io/) where a deconvolution, alignment and integration process were performed. For the annotation of the characteristics found, mass accuracy (maximum mass error 10 ppm), isotopic pattern distribution, adduct formation and elution order of the compounds based on chromatographic conditions were considered and different online public databases such as METLIN (http://metlin.scripps.edu), KEGG (http://genome.jp/kegg), HMDB (https://hmdb.ca/), PubChem (https://pubchem.ncbi.nlm.nih.gov/), ChEBI (https://www.ebi.ac.uk/chebi/) y NP Atlas (https://www.npatlas.org/). Compounds from primary metabolism were discarded in this process.The UPLC-PDA analysis was performed with an Acquity UPLC H-class , equipped with photodiode array detector (PDA), quaternary pump, on-line degasser and autosampler. Chromatographic separation was performed on a Phenomenex® Kinetex EVO C18 column , at 30 °C. The mobile phase used was water with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.40 mL/min. The elution gradient was performed as follows: 3% B for 0 to 3 min, 3 to 95% B for 3 to 30 min, 95% B for 30 to 32 min, 95 to 3% B for 32 to 35 min and 3% B for 35 to 40 min. The samples were prepared at a concentration of 1 mg/mL in MeOH LC–MS, filtered through syringe filters  and 2 μL of each sample were injected. The wavelength used was 274 nm, with spectra acquired over a range of 200–450 nm. For LCMS analysis, same chromatographic conditions were employed. A Nexera X2 LCMS Q-TOF 9030 Shimadzu® chromatograph  with electrospray ionization was used. MS data was acquired in positive mode in an P. nigrum extract as previously reported [50 value (50% inhibition of cell growth) was calculated using GraphPad Prism version 8.1.1 for Mac OS X statistics software .MTT -2,5-diphenyltetrazolium) assay was used to evaluated in vitro cytotoxicity of reported . The IC52 and treated with the IC50 or IC50/5 of P2Et or P. nigrum extract. After 6, 12, and 24 h, cells were collected and counted with 0.4% trypan blue. Using the exponential growth method (Mathusian), the population doubling time (PDT) was calculated through GraphPad Prism version 8.1.1 for Mac OS X statistics software .B16-F10 and 4T1 cells were seeded in 12-well plates at a density of 26,000 cells/cm5 cells were treated with the IC50 and IC50/2 of the P. nigrum extract (resuspended in ethanol) and IC50 of doxorubicin (resuspended in DMSO) at 0.2374 µM for 4T1 or 0.022 µM for B16-F10 for 24 h. Flow cytometry analysis was performed on a FACSAria II-U  and the data was analyzed using FlowJo v10.8.1 software (BD Life Sciences). The assays were performed in triplicate.Phosphatidylserine (PS) externalization was assessed by flow cytometry using Annexin V  and propidium iodide (PI) , as previously reported , 19. Bri2DCFDA)  as previously described [5 cells were plated and treated with the IC50 and IC50/5 of the P. nigrum extract, IC50 and IC50/5 of the P2Et extract (antioxidant control), IC50 of doxorubicin , DMSO  or ethanol  for 6, 12 and 24 h were used. Samples were acquired using an FACSAria II-U (BD Biosciences) and analyzed with FlowJo v10.8.1 software (BD Life Sciences). The assays were performed in triplicate.The production of ROS was evaluated using the probe 2′,7′-dichlorodihydrofluorescein diacetate , rotenone , or ethanol  for 6 and 12 h. After treatments, cells were incubated with 2-NBDG (Invitrogen Molecular Probes) for 30 min at 37 ℃, as previously described [1 × 10escribed . Cells w50 and IC50/5 of the P. nigrum extract and the controls for 6 h and 12 h, as previously published [The mitochondrial membrane potential of cells was evaluated using JC-1 dye  as previously reported . Brieflyublished   with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate [B16-F10 and 4T1 cells were cultured in a 37 °C and 5% COpyruvate . Abs usepyruvate .Young (6 to 12 weeks old) female C57BL/6NCrl and BALB/cAnNCrl mice were housed at the animal facilities of the Pontificia Universidad Javeriana  following the established protocols of the Ethics Committee of the Faculty of Sciences, PUJ, and National and International Legislation for Live Animal Experimentation . Each protocol was approved by the animal experimentation committee of PUJ (FUA-093–20) .P. nigrum extract and descaling up to 175 mg/kg. Lethal dose 50% (LD50) was calculated with Probit version 14 (Minitab Inc.). The dose of P2Et extract was used as previously reported [BALB/cAnNCrl and C57BL/6NCrl mice were intraperitoneally (IP) inoculated with 2000 mg/kg of 5 viable B16-F10 cells. For the breast cancer murine model, 1 × 104 viable 4T1 cells were s.c. injected into the right mammary fat pad of BALB/cAnNCrl mice as previously described [P. nigrum extract, or PBS as negative control (extract vehicle) two times per week [3) = L (major axis) x W2 (minor axis)/2 [For melanoma tumor induction, C57BL/6NCrl mice were subcutaneously (s.c.) inoculated in the right flank with 1 × 10escribed . Five daper week , 20. Thehttps://www.omiq.ai/) was used. Single live CD45 cells for each file were concatenated for analysis by opt-SNE dimensionality reduction [It was evaluated as previously reported . Brieflyeduction . Multifueduction , 21.Statistical analysis of the significance between two groups was calculated using the Mann Whitney U test. Differences among subject groups were evaluated using Kruskal Wallis and Dunn’s posttest for multiple comparisons. GraphPad Prism version 8.1.1 for Mac OS X statistics software (GraphPad Software) was used for the statistical analyses.P. nigrum at the wavelength of 254 nm showed a diversity of compounds of different polarities, observed across the different retention times. Three major peaks were observed at Rt = 15.81, 22.0, and 25.35 min, identified as piperine (3), piperolein A (6), and piperolein B (7), respectively. Compound 3 was compared with the commercial standard (STD) based on its retention time and UV spectra, unlike compounds 6 and 7, which were tentatively identified . In addition, the standardized extract of P. nigrum significantly reduced the proliferation of 4T1  or 15.67 mg/Kg (4T1 model) body weight of P.nigrum extract, which corresponds to 4 times lower doses than LD50. The P. nigrum extract significantly decreased tumor size in the 4T1 model , conventional dendritic cells (cDCs), and CD8α+ DCs, with a significant decrease in Treg, M-MDSC-LC, and PMN-MDSC-LC , as well as cDCs and CD8α+ DCs, and a decrease in PMN-MDSC-LC were also observed, but unlike what was observed in the 4T1 model, the decrease in Tregs was not significant, and a decrease in M-MDSC-LC was not observed  induced by the treatment with the -priming .Fig. 4P.P.nigrum induced a higher frequency of CD4+ T cells and a lower frequency of Treg, M-MDSC-LC and PMN-MDSC-LC Below is the link to the electronic supplementary material.


Gossypium hirustum L.) growth and development, seriously affecting lint yield and fiber quality. However, it is still unclear how P fertilizer affects fiber length.Phosphorus (P) deficiency hinders cotton  with 15 ± 0.5 mg kg−1) were applied to the plots, as the subplot, to investigate the impact of soil AP content on cotton fiber elongation and length. Therefore, a two-year (2019-2020) pool-culture experiment was conducted using the split-plot design, with two cotton cultivars  as the main plot. Three soil available phosphorus (AP) contents (P0 and P1) decreased the contents of the osmotically active solutes in the cotton fibers, including potassium ions (K+), malate, soluble sugar, and sucrose, by 2.2–10.2%, 14.4–47.3%, 8.7–24.5%, and 10.1–23.4%, respectively, inhibiting the vacuoles from facilitating fiber elongation through osmoregulation. Moreover, soil AP deficiency also reduced the activities of enzymes participated in fiber elongation (plasma membrane H+-ATPase (PM-H+-ATPase), vacuole membrane H+-ATPase (V-H+-ATPase), vacuole membrane H+-translocating inorganic pyrophosphatase (V-H+-PPase), and phosphoenolpyruvate carboxylase (PEPC)). The PM-H+-ATPase, V-H+-ATPase, V-H+-PPase, and PEPC were reduced by 8.4–33.0%, 7.0–33.8%, 14.1–38.4%, and 16.9–40.2%, respectively, inhibiting the transmembrane transport of the osmotically active solutes and acidified conditions for fiber cell wall, thus limiting the fiber elongation. Similarly, soil AP deficiency reduced the fiber length by 0.6–3.0 mm, mainly due to the 3.8–16.3% reduction of the maximum velocity of fiber elongation (VLmax). Additionally, the upper fruiting branch positions (FB10–11) had higher VLmax and longer fiber lengths under low soil AP. Low soil AP (P+-ATPase and V-H+-PPase activities yielded longer fibers. And the malate and soluble sugar contents and V-H+-ATPase and PEPC activities in the SCRC-28's fiber were more sensitive to soil AP deficiency in contrast to those of CCRI-79, possibly explaining the SCRC-28 fiber length sensitivity to low soil AP.Cotton fibers with higher malate content and V-H In summe value)  or the ae value) . These dThe leaves subtending cotton bolls provide 60% to 87% of carbon for the cotton bolls, seriously affecting their growth and development . Thus, t+), malate, soluble sugar, and sucrose. After entering the vacuoles through the reverse content gradient, these solutes exert osmoregulation, causing water to enter the vacuoles and fiber cells during elongation (+-ATPase (PM-H+-ATPase) pumps out H+, forming transmembrane ion gradients to provide the initial power for the transmembrane transport of osmotically active solutes. Meanwhile, the acidification of the surrounding environment facilitates cell wall expansion. Vacuolar membrane H+-ATPase (V-H+-ATPase) and H+-translocating inorganic pyrophosphatase (H+-PPase) play similar roles  and the related enzymes’ activities  of cotton fiber cells through the “subtending leaves (source) - cotton fiber (sink)” association. Determining whether these effects impact the cotton fiber length and the mechanisms involved is also important.Cotton fiber cells begin transitioning from expansion elongation (non-polar) to polar elongation at two days post-anthesis (DPA). The fiber cell elongation determines the length of cotton fibers, an important parameter in the textile industry . Vacuolaongation . The plaar roles . Low P sar roles  and the ce roots . Phosphoce roots ; however tobacco  but downbidopsis  under loTherefore, this research explored (1) elongation and length of cotton fibers in the presence of soil AP deficiency; (2) the relationship between osmotically active solute contents and linked enzymes activities during fiber elongation and leaf P content of subtending leaves; (3) the key osmotically active solutes and enzymes when soil AP is low. Our findings provide a reference for further research on improving fiber qualities in low AP soil.22.1−1, 0.86 mg kg−1, 64.4 mg kg−1, 3.1 mg kg−1, and 163.6 mg kg−1 in 2019 and 13.1 g kg−1, 0.85 mg kg−1, 63.3 mg kg−1, 3.0 mg kg−1, and 180.4 mg kg−1 in 2020. Meteorological data of the cotton growing seasons in 2019 and 2020 are presented in In Anyang (36°06′ N and 114°21′ E), Henan, China, Chinese Academy of Agricultural Sciences’ Institute of Cotton Research carried out a two-year pool-culture study from 2019 to 2020. Each pool was 3.6-m-long, 4-m-wide and 1.5-m-high. The experimental soil type is classified as Inceptisols (USDA Soil Taxonomy). Clay loam was the soil used in the experiment . Among s2.2-1 , 6 ± 0.5 mg kg-1 , and 15 ± 0.5 mg kg-1 . The soils from 20–40 cm depth in the field were chosen to develop P-deficiency in pool soil. Soil AP levels were regulated using triple superphosphate (44% P2O5) , and 50% of the fertilizer was used for basal application before sowing , while the other 50% for topdressing in the early flowering stage . Furthermore, the potassium fertilizer used was 150 kg K2O ha-1  and was applied as basal fertilizer  . The P frtilizer . We applrtilizer . Thereaf2.32-3), middle fruiting branches (FB6-7), and upper fruiting branches (FB10-11) were collected at 5, 10, 15, 17, 24, 31, 38, and 45 DPA (8:00-9:00 a.m.). The harvested cotton bolls were placed on ice, and the cotton fibers were peeled off from the cotton seeds within one hour. Thereafter, we analyzed the malate content and enzyme activity of one third (1/3) of the cotton fibers that were frozen in liquid nitrogen and held at -80°C. Another 1/3 was dried at 70°C to a constant weight to determine K+ and carbohydrate contents. The remaining 1/3 was used for fiber length measurement.During the flowering period of cotton plants, 8−10 cotton bolls of the first fruit node in the lower fruiting branches  at 769.9 nm.After crushing with a disintegrator, dried cotton fiber samples were sieved at 0.5 mm. Thereafter, K2 to obtain the crude extract. We centrifuged the crude extract at 12000×g (5 min), and the supernatant was collected and mixed with 150 μl of active carbon (100 mg ml-1). The mixture was centrifuged under the same conditions, and the supernatant was collected. Finally, 5 ml of the supernatant was transferred into a 10 ml cuvette, and 1 ml of Tris-HCl  was added and mixed. Incubate for 15 min with distilled water topped up to 25 ml. The malate contents were measured on the spectrophotometer  at 656 nm using the colorimetric method , and 20 mM MgClc method .Weighed (0.1 g) cotton fibers were placed in a 10 ml centrifuge tube with 5 ml of 80% ethanol (v/v) and incubated at 80°C for carbohydrates extraction (30 min). The extracts were centrifuged , and a 25 ml volumetric flask was used to collect the supernatant. Repetition of the extraction procedure was performed, and the obtained supernatant was topped up to 25 ml with ethanol . In accordance with Hendrix et al. (1993), we measured the soluble sugar and sucrose contents.2.5+-ATPase, and V-H+-PPase activities. Briefly, the fiber powder was extracted with 5 ml of the buffer containing 30 mM Hepes-Tris (pH 7.4), 250 mM mannitol, 3 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1.5% (w/v) polyvinylpyrrolidone (PVP) 4000, 1 mM dithiothreitol (DTT), and 0.1% (w/v) bovine serum albumin (BSA). In a centrifuge operated at 4°C, the homogenate was centrifuged  and the supernatants were stored to measure the enzyme activities. The reaction solution (1100 µl) for PEPC activity assay contained 800 μl of the reaction buffer (30 mM Hepes-Tris (pH 7.5), 10 mM MgCl2, 10 mM NaHCO3, and 0.5 mM DTT), 100 μl of crude enzyme solution, 100 µl of malate dehydrogenase , and 100 µl of 30 mM PEP. The PEPC activity was measured using the colorimetric method at 340 nm. For V-H+-ATPase activity determination, the reaction solution (500 µL) contained 400 μl of the reaction buffer (30 mM Hepes-Tris (pH 7.5), 3 mM MgSO4, 50 mM KCl, 0.5 mM NaN3, 0.125 mM (NH4)2 MoO4, and 0.125 mM Na3VO4), 50 μl of crude enzyme solution, and 50 μl of 20 mM ATP-Tris. Half-hour was spent incubating the mixed solution at 30°C. In order to terminate the reaction, 1 ml of the stop solution (5% (NH4)2MoO4: 5 M H2SO4: H2O=1: 1: 3) was added. Finally, 200 μl of chromogenic agent (0.25 g of aminophenol sulfonic acid in 100 ml of 1.5% Na2SO3 (pH 5.5) mixed with 0.5 g of Na2SO3) was added and the mixture was incubated for 20 min at 37°C. The V-H+-PPase activity was also determined using the colorimetric method at 660 nm. Its reaction solution (500 µl) contained 400 μl of the reaction buffer (30 mM Hepes-Tris (pH 7.5), 3 mM MgSO4, 50 mM KCl, 0.5 mM NaN3, and 0.125 mM (NH4)2 MoO4), 50 μl of crude enzyme solution, and 50 μl of 20 mM PP-Tris. The following measurement steps were the same as those employed to determine V-H+-ATPase activity. PM-H+-ATPase was isolated using Hu et al.’s method (2018). In brief, frozen fiber tissues were ground in an ice bath with 5 ml of cold buffer comprising 50 mM Hepes-Tris (pH 7.0), 300 mM sucrose, 8 mM EDTA, 2 mM PMSF, 1.5% (w/v) PVP 4000, 4 mM DTT, and 0.2% (w/v) BSA. In 4°C, the homogenate was centrifuged . In order to analyze enzyme activity in the supernatants, the supernatants were stored. The reaction solution (500 µL) for PEPC activity assay contained 400 μl of reaction buffer (30 mM Hepes-Tris (pH 6.5), 3 mM MgSO4, 50 mM KCl, 0.5 mM NaN3, 0.125 mM (NH4)2 MoO4), 50 μl of crude enzyme solution, and 50 µl of 20 mM ATP-Tris. The subsequent measurement processes were in accordance with those of V-H+-ATPase activity determination.2.6Boiling cotton bolls formed before 30 DPA in 0.1% (v/v) HCl separated fibers from cotton seeds . The fibFor cotton bolls formed after 30 DPA, the fibers were peeled and incubated at 60°C (30 min) and then at 40°C (2 h). The fibers were finally incubated in a standard assay chamber for 48 h, with temperature and humidity set at 20 ± 2°C and 65 ± 5%, respectively. The fiber length was then determined using a photoelectric stapler  .The fibers were obtained by ginning seed cotton from cotton bolls harvested on September 15, 2019, and September 15, 2020. After drying the cotton fiber samples to a constant weight at 35°C, the fiber length was analyzed at the Supervision, Inspection, and Test Center of Cotton Quality, Ministry of Agriculture and Rural Affairs, China.2.7In order to calculate means, standard errors, and coefficients of variation , Microsoft Excel 2007  was used. The SPSS statistical software Version 23.0  was used to conduct a variance analysis for all treatments at a 5% significance level, applying the least significant difference (LSD). In order to analyze each variable’s specific relevance, Pearson correlation coefficient was used.33.10 and P1) decreased the fiber K+ contents  compared to P2. Low soil AP had less effect at the FB10–11 compared to FB2–3 and FB6–7  than CCRI-79 (6.3%). CCRI-79 had fiber K+ contents 6.9 and 18.0% higher than SCRC-28 in P1 and P0, but 1.5% lower in P2  under low soil AP treatments (P0 and P1).The fiber malate content increased at 5−10 DPA and decreased at 10−31 DPA Figure 22, the soluble sugar content reduced by 9.1−9.6% and 16.5−20.9% for CCRI-79, in P1 and P0 at the three FBPs, respectively, over the two years. A similar change pattern was observed for SCRC-28, with an 8.7−12.4% and 16.5−24.5% decrease under the same conditions  (p<0.05) . The correlation between the P content of the leaves and the sucrose content of the fibers were all significantly positive (p<0.05) at 10 DPA for SCRC-28 and 31 DPA for CCRI-79  Figure 53.3+-ATPase  reduced the PEPC activities of fibers in the FB10–11 compared to FB2–3 and FB6–7 in SCRC-28 but had less effect on the three FBPs of CCRI-79. Obviously, the variations in the four enzymes involved in fiber elongation of SCRC-28 were greater than those of CCRI-79 under low-P-stress.The fiber PEPC activity presented a single-peaked curve during fiber elongation and reached its climax at 17 DPA Figure 93.4+-ATPase and PEPC activities (p<0.01), during fiber elongation (+-ATPase activities and V-H+-PPase were very significant (p<0.01) at 10−31 DPA in SCRC-28, and significant (p<0.05) at 10−17 DPA in CCRI-79 , LLmax), the beginning time of rapid elongation (TB), the finishing time of rapid elongation (TF), and the rapid elongation duration (TL=TF-TB) of cotton fibers were calculated using formulas (2), (3), and (4), respectively.The maximum velocity of fiber elongation (V0 and P1) decreased the VLmax, but increased the TL, ultimately reducing the fiber length. The fiber length variation was consistent for CCRI-79 and SCRC-28 over the two years  (p<0.01) and AP (p<0.01) had the greatest impacts on fiber length (The CVs of fiber length were lower in FBr length Table 2.3.6p<0.05) with the contents of all osmotically active solutes  in SCRC-28 but correlated only with malate contents in CCRI-79 (p<0.01) (p<0.01) with the V-H+-ATPase and V-H+-PPase activities in CCRI-79 (p<0.05)    and the  leaves) . In the ed to P2 Figure 1us study . However lettuce  and gras lettuce  due to ted to P2 . Compareed to P2 . Low soied to P2 , furtherongation . The K+,0−31 DPA .+-ATPase activities (+-ATPase declined by 13.4 and 19.5% under P1 and P0 (soil AP deficiency), respectively, compared to P2 (p<0.01) was found between the fiber PM-H+-ATPase activity and the P content of the subtending leaves during the period of 10–31 DPA  of the VLmax were greater than twice that of TL (2.8%)  and related enzymes engaged in fiber elongation  were lower at FB10–11 than at FB2–3.After analyzing the two-year VL (2.8%) Table 2,%)  in both cultivars. Compared with P2, in P1 and P0 the PEPC activity in fibers reduced by 16.6–23.4% for CCRI-79 and 20.9–40.2% for SCRC-28, respectively, over the course of the three FBPs and two years. And the above results revealed that the SCRC-28’s V-H+-ATPase and PEPC activities had higher sensitivity to low soil AP levels, explaining the sensitivity of the fiber length of the low-P sensitive cultivars to soil AP deficiency.In the experiment, the fibers’ V-H50 and P1) inhibited the fiber cell elongation leading to reduced VLmax and fiber length, mainly due to lower malate content and V-H+-ATPase and V-H+-PPase activities.Low soil AP levels  and the activities of the related enzymes  involved in fiber elongation was lower at FB10–11 than FB2–3 under soil AP deficiency , suggesting that the longer fiber lengths on the upper FBPs (FB10–11) could adapt to low soil AP compared to lower FBPs (FB2–3).Reduced osmotically active solute contents (K+-ATPase and PEPC activities of SCRC-28 were more affected strongly by subtending leaves’ P content, which may elucidate that SCRC-28 shows greater sensitivity to soil AP deficiency.Compared to CCRI-79, the fiber malate and soluble sugar contents and V-HAnalyzing the impact of soil low AP on fiber osmoregulation is beneficial for developing low-P-tolerant cotton cultivars. Furthermore, it is necessary to investigate the effects of low-P on fiber gene expression and protein synthesis.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.MS: Conceptualization, Funding acquisition, Investigation, Methodology, Writing – original draft. CZ: Conceptualization, Investigation, Writing – original draft. WF: Formal Analysis, Writing – original draft. JS: Formal Analysis, Writing – original draft. CP: Writing – review & editing. PL: Conceptualization, Writing – review & editing. HD: Conceptualization, Writing – review & editing, Funding acquisition.