Mechanistic investigations reveal that emission enhancement is attributable to strong coupling between lanthanides and surface molecules, which enables fast triplet generation (<100 ps) and subsequent near-unity triplet transfer efficiency from surface ligands to lanthanides. Moreover, the energy relay approach supports long-distance energy transfer and allows upconversion modulation in microstructures. These findings enhance fundamental understanding of energy transfer at molecule-nanoparticle interfaces and open exciting avenues for developing hybrid, high-performance optical materials.Exploration of upconversion luminescence from lanthanide emitters through energy migration has profound implications for fundamental research and technology development. However, energy migration-mediated upconversion requires stringent experimental conditions, such as high power excitation and special migratory ions in the host lattice, imposing selection constraints on lanthanide emitters. Here we demonstrate photon upconversion of diverse lanthanide emitters by harnessing triplet exciton-mediated energy relay. Compared with gadolinium-based systems, this energy relay is less dependent on excitation power and enhances the emission intensity of Tb Photon upconversion in lanthanide-doped nanoparticles enables important technological developments. Here the authors demonstrate a mechanism leading to enhanced upconversion emission in core-shell nanoparticles, and long-distance energy transfer between nanoparticles, through triplet state population of an organic surface ligand. These nanoparticles hold great promise in optical imaging9, sensing14, optogenetics17, lasing19, anticounterfeiting21, volumetric display22, and therapeutics26. Despite enticing prospects, photon upconversion has been largely limited to a small set of lanthanide activators  with ladder-like energy levels32. To this end, considerable effort has been devoted to strategies for lanthanide emitters without long-lived intermediate energy levels37. Specifically, upconversion luminescence from Tb3+ or Eu3+ ions has been achieved through cooperative sensitization upconversion41 or dimerization sensitization43. However, these upconversion processes often suffer from low conversion efficiency and weak emission. Alternatively, energy migration-mediated upconversion has proven effective for activators without long-lived intermediate energy states. Despite these successes, energy migration upconversion requires a special host lattice, such as Gd3+, capable of migrating excitation energy33. Moreover, Gd3+ excitation through a five-photon process via a Yb3+–Tm3+ pair requires a relatively high power density, and the excitation energy has a limited diffusion distance, imposing constraints for photon upconversion in large particles.Optical spectroscopy has witnessed a rapid development of lanthanide-doped nanoparticle probes over the past decade owing to their outstanding features, including sharp emission bandwidths, large anti-Stokes shifts, and high photostability, as well as the ability to convert near-infrared (NIR) excitation to short-wavelength emissionHere we demonstrate that it is possible to achieve enhanced upconversion emission through triplet exciton-mediated energy relay Fig. . Excitat4@NaYbF4:Tm@NaYF4 multilayer, core-shell nanoparticles as a model platform due to their documented high upconversion efficiency44. In a typical study, ligand-free core-shell nanoparticles were first synthesized according to a modified literature method45. Subsequently, a thin layer of activators (Tb3+or Eu3+) was coated onto the nanoparticles through cation exchange using TbCl3 or EuCl3 as the source of activators46. Finally, three molecules, namely 9-[3-carboxyl-4-(diphenylphosphinoyl)phenyl]-9H-carbazole (CPPOA), 2,2′-bis(methylene)bis(oxy)dibenzoic acid (TPDA) and 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid (TDA), were conjugated to core-shell particles  diode laser, ligand-free particles showed blue emission corresponding to 1I6 → 3H6 and 3F4 (290 and 345 nm), 1D2 → 3H6 and 3F4 (360 and 450 nm), 1G4 → 3H6 (475 nm), and 1G4 → 3F4 (650 nm) optical transitions of Tm3+ ions. No Tb3+ emission was detected, indicating that no direct energy transfer occurs from Tm3+ to Tb3+. Upon surface modification with ligands, the particles exhibited a significant decrease in UV emissions. A new set of visible emissions also appeared, arising from 5D4 → 7F6 (491 nm), 7F5 (545 nm), 7F4 (589 nm), and 7F3 (622 nm) transitions of Tb3+ ions , 615 nm (5D0 → 7F2), and 696 nm (5D0 → 7F4). To enrich the color diversity, we doped Eu3+ in the outmost NaYF4 shell of NaYF4@NaYbF4:Tm@NaYF4 multilayer nanoparticles and then performed a cation-exchange reaction with TbCl3. After ligand modification, dual emissions from Tb3+ and Eu3+ ions were realized. By carefully varying the doping concentration of Eu3+, multicolor emission was achieved  are spatially confined in the middle shell. Compared to a random distribution of donors in the core-dominated core-shell structure, this core-shell-shell design may offer higher energy transfer efficiency because of a shorter donor–acceptor distance, as revealed by the Monte-Carlo simulation  between Tm3+ and the molecules56. To validate our hypothesis, we prepared CPPOA-modified NaYbF4:Yb/Tm (1 mol%)@NaYF4:Tb core-shell nanoparticles as a control. We observed a much higher Tb3+ emission intensity from core-shell-shell nanoparticles than from conventional core-shell nanoparticles  because their proximity results in fast triplet generation of molecules and efficient triplet energy transfer from molecules to lanthanide emitters57. To validate this hypothesis, we next performed ultrafast transient absorption spectroscopy to probe the photodynamics of excitation energy at the interface of CPPOA and Tb3+-exchanged multilayer nanoparticles. The broadband (900−1300 nm) photo-induced absorption feature of molecular triplets appears on a picosecond timescale 3•H2O, (99.9%), Y(CH3CO2)3•H2O (99.9%), Yb(CH3CO2)3•H2O (99.9%), Tm(CH3CO2)3•H2O (99.9%), Tb(CH3CO2)3•H2O (99.9%), Eu(CH3CO2)3•H2O (99.9%), oleic acid (90%), 1-octadecene (90%), sodium hydroxide , ammonium fluoride , and cyclohexane  were purchased from Sigma-Aldrich. All chemicals were used without further purification.Gd(CH3CO2)3•xH2O  was mixed with 3.0 mL of oleic acid (OA) in a 50-mL flask. The resulting mixture was heated to 150 °C in an oil bath and maintained at this temperature for 30 min, at which time 7.0 mL of 1-octadecene (ODE) were added to the flask. After the mixture was cooled to 50 °C over 30 min, a 6.0-mL methanol solution containing NH4F (1.6 mmol) and NaOH (1.0 mmol) was added to the core precursor and stirred for 30 min. After removing volatile solvents, the temperature was increased to 290 °C under an argon atmosphere. After heating for 2 h, the mixture was cooled to room temperature and washed several times with ethanol. Products were re-dispersed in 4.0 mL of cyclohexane.In a typical procedure, an aqueous solution of Y(CH4:1%Tm shell precursor was prepared by mixing an aqueous solution of Ln(CH3CO2)3•xH2O  with 3.0 mL of OA and 7.0 mL of ODE. The mixture was heated at 150 °C in an oil bath for 1 h. After cooling to 80 °C, NaYF4 core nanoparticles in 4.0 mL of cyclohexane were added to the mixture and kept at 80 °C for 30 min. Subsequently, a methanol solution of NH4F (1.6 mmol) and NaOH (1.0 mmol) was added at 50 °C and stirred for 30 min. After removing volatile solvents, the mixture was heated to 290 °C under an argon atmosphere and maintained at this temperature for 3 h. After cooling to room temperature, the products were precipitated, washed several times with ethanol, and re-dispersed in 4.0 mL of cyclohexane for further use.The NaYbF4 shell precursor was prepared by mixing 1.0 mL of Y(CH3CO2)3•xH2O solution (0.2 M) with 3.0 mL of OA and 7.0 mL of ODE. The mixture was heated at 150 °C in an oil bath for 1 h. After cooling to 80 °C, the as-prepared NaYF4@NaYbF4:1%Tm nanoparticles were added to the mixture and kept at 80 °C for 30 min. Subsequently, a methanol solution of NH4F (0.8 mmol) and NaOH (0.5 mmol) was added under intense agitation at 50 °C. After 30 min, volatile solvents were evaporated by heating at 100 °C for 15 min. The mixture was then heated at 290 °C under an argon atmosphere for 2 h. After cooling to room temperature, nanoparticle products were collected, washed several times with ethanol, and re-dispersed in 4.0 mL of cyclohexane for further use44.The NaYF58. A mixture of YCl3 , OA (5.0 mL), ethanol (5.0 mL) and NaOH (0.3 g) was stirred at room temperature. After 20 min, an aqueous solution of NH4F  was added and kept for another 30 min. The mixture was then transferred to a Teflon-lined autoclave (20 mL) and heated at 220 °C for 12 h. The precipitate was collected, washed several times with ethanol, and re-dispersed in cyclohexane (4.0 mL) for further use.The microrod synthesis was performed according to a hydrothermal method58, a mixture of sodium citrate  and YbCl3  was stirred at room temperature to form a milky suspension. After that, an aqueous solution of NaF  was added. The resulting mixture was then transferred to a Teflon-lined autoclave (20 mL) and heated at 220 °C for 12 h.In a typical procedure2O3 (5.0 mmol) was first dissolved in trifluoroacetic acid (10 mL) at 80 °C, and then dried at 100 °C. The as-prepared lanthanide trifluoroacetate powder was dissolved in water to form gadolinium precursors .In a typical experiment, Gd59. Typically, a cyclohexane dispersion of the as-prepared microrod or microplate seeds (1.0 mL) was first added to a mixture of OA (3.0 mL), ODE (3.0 mL), gadolinium trifluoroacetates (0.2 mmol), and sodium trifluoroacetate (0.36 mmol). After heating to 100 °C and degassing for 30 min, the mixture was heated under a nitrogen atmosphere to 310 °C with a 12 °C/min rate and maintained at that temperature for 30 min. The products were collected, washed several times with ethanol, and re-dispersed in cyclohexane for further use. The same protocol was adopted to synthesize hierarchical NaYbF4:Tm (1 mol%)@NaGdF4 microplates except for using the microplates as seeds.This synthesis was performed by an epitaxy growth methodg for 20 min. The resulting products were washed several times with ethanol and deionized water, and re-dispersed in deionized water.The as-prepared hydrophobic nanoparticles were precipitated by the addition of ethanol and then dispersed in an acidic ethanol solution (0.1 M HCl) by ultrasonication. Nanocrystals were collected by centrifugation at 33,481 × 46. In a typical synthetic process, the as-prepared ligand-free nanoparticles (500 μL) were mixed with different amounts of TbCl3 at room temperature. After 24 h, the nanoparticles were collected by centrifugation at 39,846 × g for 30 min and washed several times with ethanol. Finally, the products were re-dispersed in 1.0 mL of ethanol.These nanoparticles were prepared by cation-exchange reactionsAn ethanol solution of ligand-free nanoparticles (1.0 mL) was separated into two equal portions. CPPOA ligands were added to the particle solution and ultrasonicated for 1 h to ensure ligand coordination to nanoparticle surfaces. After that, excess ligands were removed by centrifugation. Finally, the products were re-dispersed in ethanol for optical measurement.33, an aqueous solution containing ligand-free nanocrystals  was mixed with polystyrene bead dispersion (5.0 μL) and butanol (200 μL) at room temperature. After 30 min, the final products were collected by centrifugation at 4427 g for 5 min, washed with H2O, and re-dispersed in deionized water.In a typical experiment2SO4. The solvent was removed in vacuo. The residue was dissolved using 50 mL of dichloromethane. After the addition of 3-mL hydrogen peroxide (30%), the reaction mixture was stirred for 4 h and extracted using sodium hydrogensulfite-saturated solution. The organic layers were dried with anhydride Na2SO4. The solvent was removed in vacuo. The product was purified by flash column chromatography to afford a white powder in 80% yield 1H-NMR : δ = 7.66–7.63 , 7.601–7.56 , 7.508 , 6.91 , 2.29 ppm ; 13C-NMR : δ = 145.2, 145.1, 135.0, 134.9, 134.8, 133.2, 132.7, 132.6, 132.2, 131.9, 131.8, 131.5, 130.5, 129.5, 129.4, 129.2, 129.1, 126.8, 126.7; 31P-NMR : δ = 28.6; ESI-MS: m/z (%): 371 (44) [M+H]+.The synthetic procedure is shown in Supplementary Fig. 2SO4. The solvent was removed in vacuo. The residue was purified by flash column chromatography to afford a yellowish powder  1H-NMR : δ = 8.25 , 7.73–7.60 , 7.56 , 7.45 , 7.32–7.21 , 2.43 ppm ; 13C-NMR : δ = 145.2, 145.1, 140.7, 140.7, 140.1, 135.2, 135.1, 133.6, 132.6, 132.0, 131.9, 130.9, 129.8, 129.8, 129.7, 129.6, 129.5, 129.4, 126.9, 123.8, 123.7, 123.5, 121.0, 120.9, 110.4; 31P-NMR : δ = 28.5; ESI-MS: m/z (%): 458 (84) [M+H]+.Under an argon atmosphere, a mixture of TPPOMBr , carbazole , copper iodide , and potassium carbonate  was heated to 190 °C and stirred overnight. The reaction was then cooled to room temperature and extracted using water and dichloromethane. The organic layers were dried with anhydride Na2SO4. The solvent was removed in vacuo. The residue was recrystallized in acetone to yield white crystals  1H-NMR : δ = 13.40 , 8.27 , 8.10 , 7.99 , 7.814 , 7.71–7.66 , 7.63–7.65 , 7.484 , 7.40 ppm ; 13C-NMR : δ = 167.6, 167.6, 140.7, 140.6, 139.9, 139.3, 139.3, 136.9, 136.8, 134.7, 133.6, 132.1, 132.0, 131.9, 131.8, 131.1, 130.1, 129.0, 128.9, 128.7, 128.6, 127.7, 127.6, 127.0, 123.7, 121.1, 121.1, 110.3; 31P-NMR : δ = 28.8; ESI-MS: m/z (%): 486 (41) [M−H]−.CPPOM  was dissolved in 5.0 mL of pyridine and 2.0 mL of water. The solution was heated to reflux, followed by the addition of potassium permanganate . The reaction was refluxed until the purple color disappeared. The reaction was cooled to room temperature and extracted using dichloromethane (3 × 30 mL). The organic layers were dried with anhydride Na60. Anhydrous K2CO3  was added to an acetone (25.0 mL) solution containing 2,4-bis(bromomethyl)-1,3,5-trimethylbenzene , and salicylic acid . The resulting mixture was refluxed under magnetic stirring for 8 h. Afterward, water was introduced to the mixture, resulting in a white precipitate. The solid was washed with water before drying. Subsequently, the sample  was added to a mixed solution of methanol (25 mL), water (5 mL), and NaOH . The reaction mixture was refluxed for 3 h and then filtered, and HCl (1.0 M) was added to the filtrate. The precipitate was filtered and washed with water 1H-NMR  δ = 2.28 , 2.36 , 4.46 , 6.78 , 6.93 , 7.51 , 7.79 , 11.27 ; 13C-NMR  δ = 172.00, 161.20, 136.75, 135.75, 135.71, 135.55, 130.34, 129.36, 119.26, 117.17, 57.55, 19.34, 14.70.TPDA molecules were synthesized according to literature61, 0.47 g of 2,2′,2″-terpyridine (2.0 mmol), and 20 mL of CH2Cl2 were added to a solution of 1.30 g of m-chloroperbenzoic acid (7.5 mmol) in 20 mL of CH2Cl2. The mixture was stirred at room temperature for 13 h. After washing with 5% Na2CO3 solution and drying over anhydrous MgSO4, the solvent was evaporated and purified by washing with acetone to give the product . A sample product (2.17 g) was mixed with CH2Cl2 (80 mL). Then, Me3SiCN  was added to the mixture. After stirring for five min, PhCOCl  was added over 15 min and the mixture was stirred overnight at room temperature. The mixture was neutralized with 10% K2CO3 solution, and the aqueous phase was extracted with CHCl3. The combined organic phase was evaporated and the residue was crystallized from MeCN/THF for further use.The synthetic procedure for TDA molecules is shown in Supplementary Fig. 2SO4/CH3COOH  for 5 h and the reaction mixture was poured onto ice (45 mL). The product was filtered and washed with cold water and acetonitrile to yield the product  as a white solid 1H-NMR  δ = 8.18 , 8.21 , 8.65 , 8.87 , 13.29 ; 13C-NMR  δ = 166.44, 155.45, 154.58, 148.52, 139.36, 139.30, 125.53, 124.35, 122.19.Subsequently, the resulting compound (300 mg) was added to a solution containing ethanol (19.0 mL), water (4.0 mL) and KOH (594 mg). The reaction mixture was refluxed overnight, and then the solvent was evaporated under vacuum. The residue was dissolved in water (5 mL) and the pH was adjusted to 4 with aqueous HCl (1 M). The precipitate was removed by filtration and washed with cold water and acetonitrile. After that, the solid material was heated to reflux in a mixture of H3+ ion can be simplified as a point randomly distributed within a nanoparticle and serve as an individual energy donor. Each CPPOA ligand can be assumed as a point-like energy acceptor randomly distributed on the particle surface. The Monte-Carlo simulation was generated using the following criteria. First, 5000 Tm3+ ions are randomly distributed in the core of a core-shell structure or confined within the middle shell of a core-shell-shell structure, while 2000 dye molecules are randomly distributed on particle surfaces. Therefore, every Tm3+ ion can transfer the excited energy to all surface-bound dyes. As such, each Tm3+-CPPOA pair has a specific separation. The calculated FRET efficiency is the assumption of efficiencies for 2000 energy transfer events, which is defined as the probability of finding Tm3+ ions that can transfer the excitation energy to all surface-bound dye molecules. Using the same method, we can derive all the probability of finding 5000 Tm3+ donors to offer FRET in a core-shell or core-shell-shell particle. Thus, we can plot the probability of finding energy donors against the spatial distance from the particle surface.The energy transfer process is described based on a Förster resonance energy transfer (FRET) mechanism. Monte-Carlo calculations of energy transfer efficiency were executed using Mathematica 12 and performed in a simulated 3D coordinate matrix. In CPPOA-modified nanoparticles, each Tm62. We chose M06-2X functional in combination with 6-311G** basis set64. No imaginary frequency was detected for these optimized molecular structures. Time-dependent density functional theory (TD-DFT) calculations were performed to obtain S1 and T1 energy states of the three molecules. For excited-state calculations, we employed the same functional and basis set as ground-state calculations.Density functional theory (DFT) calculations were performed for geometry optimization and harmonic frequency analysis of CPPOA, TPDA and TDA molecules using Gaussian 16 (A.03) program1H-NMR, 13C-NMR, 31P-NMR, and high-resolution mass spectra for the 2-(Diphenylphosphoryl)-5-bromotoluene (TPPOMBr) were shown in Supplementary Figs. 1H-NMR, 13C-NMR, 31P-NMR, and high-resolution mass spectra for 4-(diphenylphosphoryl)-3-methyl-N-carbazole (CPPOM) were shown in Supplementary Figs. 1H-NMR, 13C-NMR, 31P-NMR, and high-resolution mass spectra for 9-[3-carboxyl-4-(diphenylphosphinoyl)phenyl]-9H-carbazole (CPPOA) were shown in Supplementary Figs. 1H-NMR and 13C-NMR for the 2,2′-bis(methylene)bis(oxy)dibenzoic acid (TPDA) were shown in Supplementary Figs. 1H-NMR, 13C-NMR and high-resolution mass spectra for 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid (TDA) were shown in Supplementary Figs. The structures of as-synthesized molecules were confirmed by nuclear magnetic resonance (NMR) and high-resolution mass spectroscopy λ = 1.5406 Å). Low-resolution transmission electron microscopy (TEM) was performed on a transmission electron microscope (JEOL-1400) operating at an acceleration voltage of 120 kV. High-resolution TEM images were taken using a transmission electron microscope (JEOL-JEM 2010) operated at an acceleration voltage of 200 kV. Energy-dispersive X-ray (EDX) spectroscopic analysis was performed using an energy system (Oxford INCA) operated at 200 kV. UV-Vis spectra were recorded on a UV spectrophotometer (Shimadzu 2450). Fourier transform infrared (FT-IR) spectroscopy employed an FT-IR spectrometer (Varian 3100). Photoluminescence spectra were recorded in a fluorescence spectrophotometer (Edinburgh FSP920) equipped with a photomultiplier (PMT) in conjunction with a 980-nm diode laser and a xenon arc lamp (Xe900). The lanthanide concentration was determined by a Perkin Elmer Avio 500 inductively coupled plasma optical emission spectrometer (ICP-OES).Powder X-ray diffraction (XRD) data were measured with an X-ray diffractometer (Bruker D8 Advance) with graphite monochromic CuKα radiation . Pump pulses (355 nm) were generated through a TOPAS-Prime amplifier (Light Conversion), which was pumped by a 790-nm pulse laser from a regenerative Ti:Sapphire amplifier system . The broadband probe pulse (800–1550 nm) was obtained by introducing a 790-nm laser pulse into a YAG crystal. The pump-probe delay was computer-controlled with a piezoelectric translation stage. The time resolution of laser pulses was approximately 200 fs. Nanosecond time measurements were performed with an electronically controlled delay.η = (τdonor only − τdonor–acceptor)/τdonor only. Note that τdonor only refers to the triplet lifetime of the donor in the absence of acceptors, while τdonor–acceptor represents the triplet lifetime of the donor in the presence of acceptors.Samples were excited with a pump pulse and probed at different delay times using a broadband probe pulse. A differential transmission (ΔT/T) signal in transient absorption spectra was recorded over short pulses (500 fs to 6 ns) with a probe covering  and long (1 ns to 1 ms) time delays with a probe pulse covering . To identify different components from transient absorption data, a genetic algorithm analysis was also used to distinguish different spectral species and corresponding kinetics. Triplet energy transfer efficiency can be calculated by Supplementary Information


Each carboxylate group of the 2-nitrobenzoate ligand is coordinated by CuII atom in a monodentate fashion and two TMEDA ligand nitrogen atoms are coordinate by the metal center, giving rise to a distorted square-planar coordination environment. In the crystal, metal complexes are linked by centrosymmetric C—H⋯O hydrogen bonds, forming ribbons via a R22(10) ring motif. These ribbons are linked by further C—H⋯O hydrogen bonds, leading to two-dimensional hydrogen-bonded arrays parallel to the bc plane. Weak π–π stacking interactions provide additional stabilization of the crystal structure. Hirshfeld surface analysis, normd and two-dimensional fingerprint plots were examined to verify the contributions of the different intermolecular contacts within the supramolecular structure. The major interactions of the complex are O⋯H/H⋯O (44.9%), H⋯H (34%) and C⋯H (14.5%).The reaction of copper(II) sulfatepentahydrate with 2-nitrobenzoic acid and  The CuII atom has a distorted square-planar geometry with one oxygen atom each from two nitrobenzoic acid ligands and two TMEDA ligand nitrogen atoms  acetate reacts with 2-nitrobenzoic acid and s Figs. 1 and 2 ▸.A⋯O3i and C9—H9⋯O4ii; symmetry codes as in Table 2c–axis direction, forming supramolecular ribbons linked via R22(10) ring motifs. Adjacent ribbons are connected by C3—H3A⋯O3 hydrogen bonds; these interactions lead to the formation of layers lying parallel to the bc plane. The three-dimensional network is stabilized by π–π stacking interactions with a centroid-to-centroid distance Cg1⋯Cg1iii of 3.741 (2) Å, where Cg1 is the centroid of the C5–C10 ring .The crystal packing of the title complex Fig. 2 featureset al., 2016catena-[bis[N-(2-aminoethyl)-3-amino-1-propanol]dicopper(II)] [N-pyridin-2-amine-N1]]dicopper(II) tetrahydrate [N-(pyridin-2-yl-N)pyridin-2-amine-N1]]dicopper(II) biphenyl-2,2′-dicarboxylic acid solvate monohydrate copper(II)  and the molecular electrostatic potentials (–0.2173 to 0.1248). In Fig. 3a, the red spots correspond to the O⋯H contacts. The electrostatic potential  shows donor (red) and acceptor (blue) regions. O⋯H/H⋯O (44.9%) contacts, seen as a pair of spikes of scattered points in the fingerprint plot, make the largest contribution to the total Hirshfeld surface in [Cu(2-nitrobenzoate)2(tmeda)]  points related to the H atoms. Two symmetrical wings on the left and right sides are shown in the graph of C⋯H/H⋯C interactions (14.5%). The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of O⋯H, H⋯H and C⋯H interactions suggest that van der Waals interactions and hydrogen bonding play the major role in the crystal packing.Hirshfeld surface analysis and the associated two-dimensional fingerprint plots ] Fig. 4. The sec4·5H2O  under stirring. Tetramethylethylenediamine  was added and the color changed from light blue to violet. The mixture was filtered and the filtrate was allowed to stand for slow evaporation. Single crystals suitable for X-ray were obtained after several days.An aqueous solution of sodium 2-nitrobenzoate  was added to an aqueous solution of CuSOUiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) otherwise.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021002802/zn2005sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021002802/zn2005Isup2.hklStructure factors: contains datablock(s) I. DOI: 1910225CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The bifurcated σ-hole···σ-hole stacking interactions between organosulfur molecules, which are key components of organic optical and electronic materials, were investigated by using a combined method of the Cambridge Structural Database search and quantum chemical calculation. Due to the geometric constraints, the binding energy of one bifurcated σ-hole···σ-hole stacking interaction is in general smaller than the sum of the binding energies of two free monofurcated σ-hole···σ-hole stacking interactions. The bifurcated σ-hole···σ-hole stacking interactions are still of the dispersion-dominated noncovalent interactions. However, in contrast to the linear monofurcated σ-hole···σ-hole stacking interaction, the contribution of the electrostatic energy to the total attractive interaction energy increases significantly and the dispersion component of the total attractive interaction energy decreases significantly for the bifurcated σ-hole···σ-hole stacking interaction. Another important finding of this study is that the low-cost spin-component scaled zeroth-order symmetry-adapted perturbation theory performs perfectly in the study of the bifurcated σ-hole···σ-hole stacking interactions. This work will provide valuable information for the design and synthesis of novel organic optical and electronic materials. The selAccurate calculations of the weakly bound complexes are still great challenges in the field of noncovalent interactions. It is very common that many computational methods which perform excellently on the testing weakly bound complexes fail to describe the noncovalent interactions in a new system. Thus, it is necessary to analyze the reliability and feasibility of the computational methods before the research has been conducted. Based on our previous studies on a series of π-stacked complexes ,23, the The small 2-fluorothiophene dimer was selected as a model to explore the reliability of the PBE0-D3(BJ)/def2-TZVPP calculations. As shown in Throughout the paper, we used UPICAY-M, WEPGON-M, XEGNAA-M and XEGNEE-M to represent the corresponding monomers in the crystal structures , and UPIThe energy decomposition analysis of the bifurcated σ-hole···σ-hole stacking interaction was performed by using the spin-component scaled zeroth-order symmetry-adapted perturbation theory (SCS-SAPT0) with the basis set aug-cc-pVDZ ,32,33,34The molecular electrostatic potentials and interaction energies were calculated with the GAUSSIAN 09 program package ; SCS-SAPThe two interacting conjugated backbones in each of the dimers in Eelst), exchange repulsion energy (Eexch), induction energy (Eind), and dispersion energy (Escs-disp). Additionally listed in Edisp(2)] was also calculated to approximately assess the reliability of the SCS-SAPT0/aug-cc-pVDZ dispersion energy. The values of Edisp(2) for the dimers UPICAY-D, WEPGON-D, XEGNAA-D and XEGNEE-D are −7.40, −7.52, −12.14 and −9.00 kcal/mol, respectively. Correspondingly the SCS-SAPT0/aug-cc-pVDZ dispersion energies for the dimers UPICAY-D, WEPGON-D, XEGNAA-D and XEGNEE-D are −7.50, −7.73, −12.49 and −9.43 kcal/mol, respectively. Clearly the dispersion energies calculated by two different methods are quite close to each other. All these results show that the SCS-SAPT0/aug-cc-pVDZ calculations are reliable for the study of the bifurcated σ-hole···σ-hole stacking interactions in the dimers UPICAY-D, WEPGON-D, XEGNAA-D and XEGNEE-D.As shown in The AIM analyses have been carried out to further uncover the bonding characteristic of the bifurcated σ-hole···σ-hole stacking interaction. ρb are all low and the values for ▽2ρb are all larger than zero at the bond critical points of the S···S contacts in the four dimers, which exhibits the characteristics of the existence of noncovalent interactions. The bond paths in 2ρ) is the sum of three eigenvalues of the Hessian matrix  and ellipticity (ε) can be calculated as λ1/λ2−1. Besides the π-character of the bonding, the ellipticity is also a measure of the structural stability [It is clear in tability . A previtability . In TablIn this work, the bifurcated σ-hole···σ-hole stacking interactions between organosulfur molecules were studied in detail with a combined method of the Cambridge Structural Database search and quantum chemical calculation. The organosulfur molecules selected in this work are important building blocks for organic optical and electronic materials. It was found that the binding energy of one bifurcated σ-hole···σ-hole stacking interaction is in general smaller than the sum of the binding energies of two free monofurcated σ-hole···σ-hole stacking interactions. The main reason is that the monofurcated σ-hole···σ-hole stacking interactions in the bifurcated σ-hole···σ-hole stacking interaction are subject to some geometrical restrictions and not in their optimal structures. The results of energy-component analyses for the total interaction energies of the dimers UPICAY-D, WEPGON-D, XEGNAA-D and XEGNEE-D show that the bifurcated σ-hole···σ-hole stacking interactions are still dispersion dominated, although the contributions of the electrostatic energies to the total attractive interaction energies increase significantly and dispersion energy contributions decrease significantly in comparison with the linear monofurcated σ-hole···σ-hole stacking interactions.The calculations of the 2-fluorothiophene dimer which is taken as a model show that the PBE0-D3(BJ)/def2-TZVPP performs perfectly for the study of the complex bound by the σ-hole···σ-hole stacking interaction, and the accuracy of the PBE0-D3(BJ)/def2-TZVPP method is comparable to the accuracy of the “gold-standard” CCSD(T)/CBS method. Another important finding of this study is that the PBE0-D3(BJ)/def2-TZVPP interaction energies for the dimers UPICAY-D, WEPGON-D, XEGNAA-D and XEGNEE-D are almost the same as the corresponding SCS-SAPT0/aug-cc-pVDZ interaction energies. The calculations of the dispersion energies prove the reliability of the energy components calculated by the SCS-SAPT0/aug-cc-pVDZ method. The SCS-SAPT0/aug-cc-pVDZ calculation is very cheap and it can be used for the study of very large weakly bound complexes.Besides the bifurcated σ-hole···σ-hole stacking interactions between C–S σ bonds, bifurcated σ-hole···σ-hole stacking interactions may exist between other σ bonds. Such kinds of bifurcated σ-hole···σ-hole stacking interactions are under investigation in our laboratory.


Ircinia mutans Wilson (Irciniidae) is a sponge with antimicrobial and cytotoxic constituents.I. mutans.Our objective was to characterise the cytotoxic constituents of two seasonal collections of The sponges were extracted in methanol-dichloromethane and their constituents were purified and characterised using column chromatography, GC-MS, 1 D and 2 D NMR. Anti-proliferative activities of the compounds, were evaluated using 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay  against leukaemia (MOLT-4), breast (MCF-7) and colon cancer (HT-29) human cells.1), ent-furodysinin (2) and furoircin (3) and ten sterols were characterised in I. mutans, for the first time. Cholesterol (4), cholesta-5, 7-dien-3β-ol (5) and ergosterol (6) were determined in the sponge from the winter collections, while cholesta-5, 22-dien-3β-ol (7), 24-methyldesmosterol (8), campesterol (9), stigmasterol (10), γ-ergostenol (11), chondrillasterol (12) and γ-sitosterol (13) were detected in the summer samples. The steroids from the winter collection exhibited cytotoxic activity with IC50 values of 13.0 ± 0.9, 11.1 ± 1.7 and 1.1 ± 0.4 µg/mL, against the mentioned cancer cell lines, respectively, while those from the summer sample, showed greater activity, IC50 = 1.1 ± 0.2 μg/mL against MOLT-4. The purified steroids showed potent MOLT-4 cytotoxic activity, IC50 values = 2.3–7.8 µg/mL.Three furanosesquiterpoids; furodysin (I. mutans is a rich source of cytotoxic steroids, and introduces 3 as new natural product. Considering the high cytotoxic activity of the steroids, these structures could be candidates for anticancer drug development in future research.The present study suggests that  Ircinia Nardo (Irciniidae) comprises about 73 species which have been widely investigated for drug discovery. This genus of sponge is a rich source of a variety of steroids, terpenoids and other types of secondary metabolites with different biological activities (Faulkner Sponges (Phylum Porifera) are the most primitive multicellular eukaryotic organisms. They are sessile without defense organs and therefore produce different secondary metabolites to protect themselves from predators and pathogens in their environment Proksch . The genFaulkner .Axinella sinoxea Alvarez & Hooper (Axinellidae), resulted in the identification of eight steroids including: cholesta-5,22-dien-3β-ol, cholest-5-en-3β-ol , ergosta-5,22-dien-3β-ol, ergost-5-en-3β-ol (9), stigmasta-5,22-dien-3β-ol , γ-sitosterol , 33-norgorgosta-5,24(28)-dien-3β-ol and stigmasta-5,24(28)-dien-3β-ol. The cytotoxic activity of the compounds was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay against three human cancer cell lines MOLT-4, MCF-7 and HT-29, and showed that the steroids are potent cytotoxic agents with IC50< 10 µg/mL -5α,6α-epoxy-24-ethyl-cholesta-7-en-3β,9α-diol and 5α,6α-epoxycholesta-7-en-3β,9α-diol have cytotoxic activity against three human cancer cell lines, MCF-7, Hep-G2 and LU-1.Steroids are the important class of metabolites that have two main biological functions: structural roles, such as with cholesterol which is an important component of cell membrane, and physiological roles such as signalling molecules that activate steroid hormone receptors , which have shown ichthyotoxicity against killifish Oryzias latipes (IC50 15 μg/mL and 20 μg/mL respectively), and the latter has also shown cytotoxicity against B-16 melanoma cells in vitro (IC50 33 μg/mL)  in vitro but inactive against two others N. dubius Baylis (Heligmosomidae) and Hypselodoris nana (Chromodorididae) in vivo test above 1000 ppm  and Dysidea fragilis Montagu (Dysideidae) , and molluscs , showed that concentrations of bromo-alkaloid compounds in ectosome layer during the summer are more than those in the winter season and that the levels of secondary compounds from the outer layer of the sponge, were significantly correlated with water temperature. Abdo et al.  and found environmental and physiological factors to be important in the production of this compound. They showed that “salicylihalamide A” concentration was significantly correlated with water temperature and was significantly higher in summer than in the winter collections in sponges from Bremer Bay. Turon et al.  was used as an internal standard for referencing 1H and 13C NMR spectra. Data acquisition and processing was accomplished using Bruker Topspin 2.1. EI-MS spectra were recorded on an Agilent 5975 C inert GC/MS instrument. For isolation and purification of the sponge’s metabolites, different chromatographic separations were performed, including silica gel open column chromatography (CC: 0.063–0.200 mm particle size), flash column chromatography (FCC: 0.040–0.063 mm particle size) and TLC using silica gel 60 F254 pre-coated plates (0.25 mm film thickness). The adsorbents were purchased from Merck, Darmstadt, Germany. For further purification of the fractions, reversed-phase (RP-18) HPLC analyses were performed using a Knauer semi-preparative HPLC with a K-1050 pump and a four wavelength K-2600 UV detector set at λ 210 nm , operating at resonance frequencies of 500 MHz for Ircinia mutans was collected by scuba diving in January 2015, and June 2016 at a depth of 10–13 m near Larak Island in the Persian Gulf. Samples were placed immediately in plastic bags containing seawater and transferred to the laboratory on ice and then stored at −20 °C after other organisms, such as brittle sea star, bivalves, barnacles, oligo- and poly-chaetes, were removed. One of us, M. Nazemi, the marine biologist, identified the sponge sample, based on scanning and optical microscope studies of skeletal slides and dissociated spicule mounts with the sponge taxonomic keys developed by John N.A. Hooper (. Hooper . Parts on-hexane (3 × 250 mL), DCM (3 × 250 mL), and 1-butanol (3 × 200 mL) to afford three fractions: n-hexane (5.05 g), DCM (1.92 g), and 1-butanol after evaporation of the solvents in vacuum. On the basis of TLC examination, we choose the hexane fraction for further phytochemical investigation.The sponge sample collected in winter (W) (652 g fresh weight) was cut into small pieces (approximately 1 cm) and extracted by methanol (2 × 4 L) for 4 days followed by the same volumes of dichloromethane (DCM) continuously at room temperature in the dark to afford residues of MeOH (3.2 g) and DCM (1.4 g) extracts after evaporation of the solvents in reduced pressure at 40 °C. The fresh sponge sample (3.29 kg) collected in summer (S) was extracted by MeOH and DCM as described earlier  and then to pure methanol to afford 24 fractions. Compound 1 (240 mg) was obtained as a colourless oil and detected as a UV quenching spot (fR = 0.5) on silica gel F254 TLC using 100% n-hexane.The MeOH and DCM extracts of the winter samples were mixed based on their similar patterns from the TLC analyses. The combined extracts were subjected to silica gel-FCC . The column was eluted with n-hexane: EtOAc (9: 1); FW9 and 10 (118 mg) were pooled based on their compositional similarity. After TLC analyses, they were subjected to FCC (40 × 2 cm) over silica gel  using different portions of hexane, DCM and methanol for further purification. The resulting fractions F51-80  were pooled and dried under reduced pressure. The silica gel TLC using DCM: MeOH  of FW9-10-F51-80 exhibited a single UV-quenching spot. The isolated compound was subjected to 1H and 13C NMR spectroscopy and GC-MS analyses and found to contain at least three major steroids 4–6.The fractions eluted by 3-impregnated (7.5 g) silica gel (150 g). The column was eluted with different portions of n-hexane and DCM followed by MeOH to afford 32 fractions. Compound 1–3 were detected on TLC in the hexane rich fractions as described above and were further purified with RP18 semi preparative HPLC with the retention times of 7.48, 7.75 and 8.42 min, respectively (The hexane layer of the methanol extract of the summer-sponge sample was subjected to FCC (50 × 4.5 cm) over AgNOectively .4–13 (Repeated silica gel (45 g)-FCC (50 × 3 cm) of fractions F6-F10 (300 mg) as described for that of the winter sample yielded a pale-yellow powder (FS6-10- F25-29) after removal of the eluting solvents (98 mg). The fraction was further analysed by GC-MS for characterisation of its constituents; 4–13 . For fur4–13 . These f4–13  and furtGC-MS analyses were performed using an Agilent 7890 A GC coupled to HP-6890 mass spectrometer operating in EI mode at 70 eV. The GC was equipped with a DB-5 MS and HP-5 MS  for the winter and the summer samples, respectively. To analyse the steroids, the oven temperature was set at 265 °C for 20 min, increased to 300 °C at 5 °C/min and kept for 10 min at the final temperature. Helium (He) was used as the carrier gas with a flow rate of 1 mL/min. The injector temperature was set at 300 °C, in the split mode (1:10). The injection volume was 0.1 μL for all of the samples -trifluoroacetamide (BSTFA) containing 1% trimethylsilyl chloride (TMCS) was added to 2 mg of F9,10 -F51-80 and vortexed well. Afterwards, the mixture was heated in a hot water bath (60 °C) for 1 h. Samples were evaporated under a stream of nitrogen gas, redissolved in 200 μL DCM and the resulting solution was subjected to GC-MS as previously described  and MCF7  cells were obtained from National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. HT-29  cell line was purchased from Iranian Biological Resource Centre, Tehran, Iran. MOLT-4 and MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% v/v foetal bovine serum, penicillin (100 units/mL) and streptomycin (100 μg/mL), and HT-29 cells were cultured in DMEM medium supplemented with 10% v/v foetal bovine serum, penicillin (100 units/mL) and streptomycin (100 μg/mL) and In vitro cytotoxic activity of compounds was evaluated by using the MTT reduction assay. MTT is a rapid colorimetric assay that can be used to measure cytotoxicity, proliferation or activation of the cells (Mosmann 50 (concentration that results in 50% inhibition of cell viability) for each compound was calculated with Curve Expert statistical analyses : colourless perfumed oil (3.8 mg.). [α]D19 +240 , EIMS (resulting from GC-MS) m/z (rel. int.%): 216 [M]+ C15H20O (26), 201 [216 – CH3]+ (6), 147 (7), 135 (41), 133 (16), 119 (21), 115 (11), 107 (100), 105 (29), 93 (84), 91 (61), 81 (30), 79 (35), 77 (44), 67 (17), 55 (22), 53 (35), 41 (23). For 1H NMR  and 13C NMR  spectroscopic data see in The spectroscopic data of previously described compounds are presented in the supplemental material.t-test was used between the mean activity of winter and summer samples, where the assumption of equal variances was met. Significance levels in all tests was 5% (p < 0.05) and data were expressed as mean ± SD. Data were analysed using SPSS statistical software .The normality of data and the homogeneity of variances were surveyed with Kolmogorov–Smirnov and Leven’s tests, respectively, and log10 transformation of some data was necessary to achieve analysis requirement. Then, the effect of each fraction on cell line were compared by means of one-way ANOVAs, and the mean comparison was conducted with the Duncan’s test at 5% significant level for steroid’s sub fractions. To compare the effect of steroids extracted from two seasons on MOLT-4 cell line, the independent sample 1–3 , 6.24 , and δ 7.17 (s) suggested a mono-substituted furan ring in the molecule. An isopropenyl group represented by two broad singlet signals at δ 4.87 and 4.72 and an olefin methyl at δ 1.77. The signals of a tri-substituted cyclohexene moiety were similar to those reported for furodysin represented by an olefin methyl group signal at δ 1.63 which is coupled with an allylic methine at δ 5.39 , together with two methylene at δ 1.68 and 2.0 and two methine signals at δ 2.41 and 2.24. A methylene signal at δ 2.38  and 2.07  attached the cyclohexene ring to the furan ring. Using the APT 13C NMR and HSQC spectra, 15 signals representing four CH2, six CH, two CH3 and three quaternary carbon atoms suggested the structure of a sesquiterpene for compound 1. The connectivity of the proton signals to their respective attached carbon atoms were confirmed by an HSQC experiment. Three methine and one quaternary carbon signals at δ 142.5, 111.5, 139.4 and 123.8, respectively, represented the mono-substituted furan ring, the signals of the isopropenyl resonated at δ 22.8 (CH3), 110.1 (CH2) and 147.6 (C). In the HMBC spectrum the cross peaks between H-12 and C-3 and C-11 confirmed the connectivity of the furan and cyclohexene rings via C-12, and the cross peaks between H-6 and C-5 and H-15 and C-9 and C-10 determine the position of the methyl and isopropenyl group on the cyclohexene ring. Two small (J = 3.8 Hz) and one big (J = 11.9 Hz) coupling constants for H-6 at δ 2.24 and a weak cross peak between H-14 and H-12 in the NOESY spectrum was compatible with the cis configuration at C-6 and C-11 substitution  (4), cholesta-5, 7-dien-3β-ol (5) and ergosta-5, 7, 22-trien-3β-ol (ergosterol) (6) . The structures of the steroids were elucidated by GC-MS and 13C NMR spectral data analyses, which were confirmed by comparison to those previously reported in the literature for the authentic compounds. The presence of Δ5-en, Δ5,7-dien and Δ5,7,22-trien were confirmed by the 13C-NMR spectra which exhibited signals in the sp2 carbon region (δ 116.25–141.40). The chemical shifts of the 13C NMR signals of the sub-fraction FW9-10- F51-80 (4–6) are consistent with the spectroscopic data previously reported for the above mentioned compounds , ergosta-5, 24-dien-3β-ol (24-methyldesmosterol) (8), ergost-5-en-3β-ol (campesterol) (9), stigmasta-5,22-dien-3β-ol (stigmasterol) (10), γ-ergostenol (fungisterol) (11), 5α-stigmasta-7,22-dien-3β-ol (chondrillasterol) (12), and γ-sitosterol (clionasterol) (13). GC-MS identification of the sterols was done by comparing their GC relative retention index calculated on a non-polar capillary column relative to a series of normal alkane (C21-C40) and their mass spectra compared with those reported in data banks  (7 (10.2%), 5 (13.1%), 6 (49.3%), 8 (12.9%); FH3 contained compound 4 (60.8%); FH4 constituted of compounds 4 (6.3%), 9 (6.3%), 10 (45.7%), 11 (4.4%), 12 (15.9%) and finally FH5 presented compounds 13 (65.8%) as its major constituent (Five subfractions (FH1-FH5) which were collected from HPLC column of the steroidal fraction (FS6-10- F25-29), and their analysis by GC-MS showed that the variety of steroids was greater than that of the winter sample. The above sub-fractions consisted of about ten Δe Tools) . The resstituent .1 and FW9-10- F51-80 (compounds 4–6) obtained from the winter sample; and F2  and FS6-10-F25-29 (compounds 4–13) obtained from summer sample were measured against three human cancer cell lines, MOLT-4, MCF-7 and HT-29 (1) obtained from the winter sample showed weak cytotoxic activity against HT-29 cell line with an IC50 value of 69.1 ± 12.8 μg/mL (means ± S.E.M.), while the non-polar fraction  obtained from the summer sample showed moderate cytotoxic activity against all three cell lines with IC50 values 33.9 ± 2.0, 50.9 ± 1.0 and 37.8 ± 4.9 μg/mL (means ± S.E.M.), respectively  against the cell lines. We combined these three compounds again with ratios of 1:2:3, respectively, and tested against three cell lines. This combination showed weak cytotoxic activity against MOLT-4 cell lines with IC50 value of 70.0 ± 8.9 μg/mL (means ± S.E.M.).The cytotoxicity of compound nd HT-29 . Cisplatectively . After p50 values of 13.0 ± 0.9, 11.1 ± 1.7 and 1.1 ± 0.4 μg/mL against the mentioned cell lines, respectively. Steroids from summer sample FS6-10- F25-29 just was tested against MOLT-4 cell lines and showed strong activity with an IC50 value of 1.1 ± 0.2 μg/mL. The cytotoxic activity of the steroids purified from the summer sample, FH2, FH3, FH4 and FH5 were evaluated against MOLT-4 cell line and showed that all steroids are potent cytotoxic compounds with IC50 values of 7.8 ± 0.2, 3.1 ± 0.7, 2.2 ± 0.3 and 2.3 ± 1.1 μg/mL, respectively  was previously isolated from genus I. mutans. Cholesterol (4) (36.8% area), cholesta-5,7-dien-3β-ol (5) (22.6% area) and ergosta-5, 7, 22-trien-3β-ol (ergosterol) (6) (10.7% area) were the major steroids from the winter sample. The main steroids detected in summer sample like winter sample consisted of 4 (46.8%), 5 (17.3%) and 6 (9.3%). The other steroids (7–13) were the minor steroids isolated from the summer sample of this sponge which were not detected in the winter sample. Ergosterol has been isolated from most calcareous sponges such as Leucetta chagosensis Dendy (Leucettidae), Pericharax heteroraphis Poléjaeff (Leucettidae), Clathrina sp. Grey (Clathrinidae), and Grantiopsis sp. Dendy (Lelapiidae) as their major constituent, and in many other species, cholesterol is reported as the minor sterol  is located in a subtropical region, where the water temperature during winter (18 °C) is substantially lower than of the summer (29 °C), we can infer that the greater variety of isolated steroids and furanosesquiterpenes from the summer sample is related to water temperature, which of course is likely also correlated with great fouling and competition from other organisms.5 and Δ5,7 steroids, but also comprises Δ7 sterols, showed stronger cytotoxic activity  than the winter sample  against MOLT-4 cell lines (p < 0.05). Various researches showed that environmental variations can affect the bioactivity of natural compounds. Turon et al. (Crambe crambe Schmidt (Crambeidae) and showed the highest biological active in late summer and autumn, similar to the results reported here. Cholesterol with IC50 value 3.1 ± 0.7 μg/mL was one of the potent cytotoxic steroids which extracted from Persian Gulf sponge, I mutans. Kellner-Weibel et al.  leaves extract and γ-sitosterol on cell proliferation, apoptosis and cell cycle arrest in breast and lung cancer cells, and showed that γ-sitosterol with induces G2/M cell cycle arrest and apoptosis through c-Myc suppression in MCF-7 and A549 cells was a potent anticancer agent.In this research the steroids showed stronger cytotoxic activity than did the furanosesquiterpenes. Although steroids obtained from both seasons showed strong cytotoxic activity, it should be noted that the steroid fraction from the summer sample, which not only contains more diverse Δn et al.  found cll et al. , showed j et al. , investiI. mutans have strong cytotoxic activity on MOLT-4, MCF-7 and HT-29 cell lines with IC50 value <10 µg/mL. The steroids composition of the sponge, I. mutans is similar to those of its neighbouring sponge, collected from the same location, A. sinoxea. Both fractions and purified compounds have the same range of activity against the mentioned cell lines with IC50 values of 1.20–4.12 μg/mL (Jamebozorgi et al. Generally, we showed that steroids from I. mutans, is a rich source of valuable secondary metabolites. Steroids are one of the dominant groups of secondary metabolites found in I. mutans and they show strong cytotoxic activity against three human cancer cell lines. Therefore, they can be considered as candidates for further investigation as anticancer agents. Also, the study of the extracted metabolites of this sponge in the winter and summer season showed that both the diversity and cytotoxic activity of metabolite extracted in the summer was greater than winter season, However, additional sample collections and statistical analyses are needed to verify these differences in future research. Finally, compound 3 or its proper isomer may be considered as intermediates in the biosynthetic pathway of compounds 1 and 2, respectively.The Persian Gulf sponge,


The structure has been reported previously using room-temperature data. Here, a more complete description of the intermolecular interactions, including a Hirshfeld surface analysis, is reported.The structure of the product obtained from the reaction of pyridine-2,3-dicarboxylic acid and hydrated copper(II) chloride in hot aqueous NaOH solution was shown to be {[Cu 3(C6H4NO3)4(OH)2(H2O)2]n or [Cu3(μ-OH)2(μ-nicNO)4(H2O)2]n (nicNO is pyridine-3-carboxylate N-oxide), a structure obtained from room temperature data and reported previously. The present determination is improved in quality and treatment of the H atoms. A Hirshfeld surface analysis of the intermolecular interactions is presented.The product obtained from the reaction of pyridine-2,3-dicarboxylic acid and hydrated copper(II) chloride in hot aqueous NaOH solution was determined by low temperature X-ray diffraction to be [Cu N-oxidation of the pyridine ring can significantly increase its electron-donating ability because the charge-polarized pyridine-N-oxide moiety can donate three pairs of electrons while a neutral nitrogen atom in pyridine only gives one pair of electrons. Therefore, it is expected that N-oxidation can increase the coordination capacities and flexibility of the ligand. Metal complexes of pyridine-N-oxide ligands have been found to be particularly useful in the selective adsorption and separation of gases (CO2 over CH4) and as anti-HIV and luminescent agents  in the crystal lattice 2(H2O)2(μ-nicNO)4]n (1) (nicNO is pyridine-3-carboxylate N-oxide) as the unexpected product from the reaction of pyridine-2,3-dicarboxylic acid with hydrated CuII chloride. It appears that oxidation and decarboxylation of the starting acid occurred during the reaction, as has been seen previously  vs 0.0416 for 2525 with I > 3σ(I) in NICTCU and 0.0538 for 3349 with I > 2σ(I) in NICTCU01. The present structure has slightly better s.u.’s on all derived parameters than obtained for NICTCU and significantly better ones than those obtained by Kang et al.. One deficiency of the NICTCU structure is the free refinement of hydrogen-atom parameters, a risky procedure with room-temperature data when heavy atoms are present, which led to C—H distances for the aromatic rings varying from 0.97 (2) to 0.84 (3) Å and O—H distances of 0.77 (3) to 0.41 (4) Å, the last three being particularly unrealistic. In addition, there was no absorption correction despite a linear absorption coefficient of 2.422 mm−1. Kang et al. performed an absorption correction and treated hydrogen atoms appropriately, but with an Rint of 0.0780 their data are clearly of poorer quality than in the present case (Rint = 0.0208).N-oxide atom from the bridging nicotinato-N-oxido ligand on each end copper atom (O3ii and O3iii) is shown in Fig. 1N-oxide atom from the bridging nicotinato-N-oxido ligand (O3ii) in the apical site and the basal sites occupied by the bridging hydroxide (O7—H7) and the water molecule (O8) in trans positions, and a carboxylate oxygen atom from the bridging nicotinato-N-oxido ligand (O1) and the bridging nicotinato-N-oxide ligand (O5i). The Cu1—O distances and bond angles are in line with those typically seen for tetragonally elongated, square-pyramidal CuII. Cu2 is coordinated by the bridging hydroxide (O7—H7) and a carboxylate oxygen of the nicotinato-N-oxide ligand (O4) and their symmetry-related counterparts. Although rigorously planar, the coordination about Cu2 shows a rhombic distortion from square geometry due to the difference in the Cu2—O4 [1.9687 (11) Å] and Cu2—O7 [1.9240 (11) Å] bond lengths and the O4—Cu2—O7 angle of 87.69 (5)°. This geometry is quite comparable to those in the previously reported structures  to Cu2 with the Cu2—O3ii distance of 2.6828 (15) Å being considerably longer than the Cu1—O3ii distance [2.4208 (13) Å] but definitely shorter than the sum of the van der Waals radii (2.92 Å), indicating a short contact. The O7—Cu2—O3ii and O7i—Cu2—O3ii angles of 81.66 (5) and 98.34 (5)°, which differ greatly from 90°, suggest the coordination of Cu2 should not be described as an elongated octahedron. There are close to 100 structures listed in the CSD 2(H2O)2(μ-nicNO)4], are connected into chains extending along the c-axis direction by coordination of N-oxide oxygen atom O3 to atom Cu1i of the next unit  x + 1, y + 1, z) as well as a ii, Cu2ii, Cu1iv, O8iv and O6i  Fig. 3. The O8—d Table 2 is part et al., 2016) using the fragments 2-, 3- and 4-carboxypyridine-N-oxide yielded 20 hits, of which 16 were complexes of 4-carboxypyridine-N-oxide, three contained 3-carboxypyridine-N-oxide, including the prior report of the title compound, and one contained 2-carboxypyridine-N-oxide. The last (3-carboxypyridine-N-oxide)(squarate)]n 4(H2O)2]n  where the substantially flat regions of the plot again occur over the pyridine rings. Parsing the intermolecular interactions into specific types is accomplished with the fingerprint plots , H⋯C/C⋯H  and O⋯Cu  interactions. Not shown are the C⋯C (7.9%) and H⋯N/N⋯H (2.5%) contacts, with the former corresponding primarily to the slipped π-stacking interactions.An effective means of probing intermolecular interactions is Hirshfeld surface analysis  was added to an aqueous solution (3.5 mL) containing pyridine-2,3-dicarboxylic acid  and NaOH , the mixture was stirred at 333 K for 2 h and then cooled to room temperature. After standing for a week, the light-blue precipitate that formed was filtered off and dried. Dark-blue, block-like crystals were obtained by slow evaporation of a solution of the precipitate in 5 mL of distilled water at room temperature. (yield: 30.61% based on Cu). Analysis calculated for: C, 27.00; H, 1.94; N, 4.50%. Found: C, 26.86; H, 2.02; N, 4.46%. IR (cm−1 KBr): 445, 489, 547, 612, 674, 688, 768, 798, 948, 1019, 1044, 1130, 1225, 1376, 1408, 1441, 1564, 1594, 1619, 2994, 3043, 3069, 3355.An aqueous solution of CuClUiso(H) = 1.2Ueq(C). Those attached to oxygen were placed in locations obtained from a difference map and were refined with DFIX O—H = 0.84 (1) Å restraints.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021002000/jq2004sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989021002000/jq2004Isup2.hklStructure factors: contains datablock(s) I. DOI: 2063970CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


After more than forty years of research, substantial progress has been made in understanding this unique receptor, yet the molecular mechanism of its ligand entry pathway remains uncertain. Published structures of human σ1R reveal its homotrimeric organization of a cupin-fold β-barrel body that contains the ligand binding site, a carboxy-terminal V-shaped two-helix bundle, and a single amino-terminal transmembrane helix, while simulation studies have suggested a ligand entry pathway that is generated by conformational rearrangements of the cupin-fold domain. Here, we present multiple crystal structures, including an open-like conformation, of σ1R from Xenopus laevis. Together with functional binding analysis our data suggest that access to the σ1R ligand binding site is likely achieved by protein conformational changes that involve the carboxy-terminal two-helix bundle, rather than structural changes in the cupin-fold domain.The sigma-1 receptor (σ The nonopioid sigma-1 receptor (σ1R) is localized to the endoplasmic reticulum (ER), interacts with multiple effector proteins and various synthetic ligands, and is implicated in many diseases. Here, authors provide structural and functional evidence to reveal the likely ligand entry pathway for σ1R. It interacts with various effector proteins1, including ion channels6 and G-protein coupled receptors9, and it is implicated in many diseases, including neurodegenerative disorders12 and cancer14. Identified in 197615 and cloned in 199616, its first atomic structure was solved in 201617. The structural information, together with information gathered from functional studies, has provided insight into the key mechanistic elements of σ1R’s function18. However, critical mechanistic details of how ligands access their binding site in σ1R remain enigmatic.The sigma-1 receptor (σ1R (hσ1R) show that hσ1R adopts a homotrimeric configuration, with each protomer comprising a single transmembrane helix (α1) at the amino-terminus, followed by a cupin-fold19 β-barrel body containing the ligand-binding site, and a membrane-adjacent V-shaped two-helix bundle (α4/α5) at its carboxy-terminus covering the cupin-fold domain like a lid20  would allow a ligand to enter σ1R through structural rearrangements of the cupin-fold domain, which involves unfolding and refolding of the β barrel; the second proposed pathway (PATH2) would allow a ligand to access its binding site in σ1R through the opening between α4 and α5 . Together with mutagenesis and binding analysis, the structural data support the notion that ligand entry to σ1R is achieved through PATH2.To provide experimental evidence to assess ligand entry to σ1R, our initial strategy was to capture a σ1R structure in its open conformation. To achieve this goal, we screened expression for ten σ1R homologs from different species and crystallized well-behaved clones without adding any known ligand for structure determination. In the end, we solved a structure for the wild-type σ1R from Xenopus laevis (xlσ1R), which shares 67% sequence identity and 89% sequence homology with human σ1R (hσ1R) , except the orientation of transmembrane helix α1  provides only limited details about the shape of the electron density  mediate crystal contacts between adjacent asymmetric units  or S1RA (antagonist), directly into the xlσ1Rclosed-endo crystals, and solved both structures, xlσ1Rclosed-PRE084 and xlσ1Rclosed-S1RA, respectively. Soaking of either ligand changed little on the xlσ1R crystal packing. Both xlσ1Rclosed-PRE084 and xlσ1Rclosed-S1RA are almost identical to xlσ1Rclosed-endo with all-atom RMSDs < 0.25 Å when aligning the three structures  bound in the ligand-binding pocket  different from the xlσ1Rclosed-endo crystals (cuboid-like) were obtained and their structure was determined to 3.56 Å (xlσ1Ropen-endo). The xlσ1Ropen-endo structure was solved in the P212121 space group, with each asymmetric unit containing 12 protomers. These protomers are nearly identical to each other with all-atom RMSDs < 0.2 Å between the protomers. Similar to all σ1R structures, xlσ1Ropen-endo also assembles as homotrimers  and S1RA (~7.6 Å × 5.2 Å transversely) to pass through. Therefore, the xlσ1Ropen-endo structure may represent an open-like conformation for σ1R and is consistent with PATH2 that predicts the ligand entry between α4 and α5.Structural comparison between xlσndo Fig. . Structu418 Fig. . The othain Fig. , thus crain Fig. . Such anain Fig. . This opain Fig. , which i1Rclosed-endo, the xlσ1Ropen-endo crystals were grown in the absence of any known ligand, but the xlσ1Ropen-endo structure also has an unidentifiable electron density within its ligand-binding pocket  or soaking PRE084 directly into the xlσ1Ropen-endo crystals . As expected, both xlσ1Ropen-PRE084-co and xlσ1Ropen-PRE084-soak structures are nearly identical to xlσ1Ropen-endo , which can be evaluated by stoichiometry measurement using isothermal titration calorimetry (ITC). After introducing a pair of cysteine mutations at residue positions 179 and 203 (xlσ1R-C179/C203), the formation of the disulfide bond was catalyzed by oxidation. All ITC isotherms measured in this study were best fitted by a one-site model. As expected, the number of the available binding sites per protomer (stoichiometry) in xlσ1R-C179/C203 decreased to 0.18 ± 0.02 after oxidation compared to 0.92 ± 0.01 before oxidation  of xlσ1R-C179/C203 for PRE084 also increased by ~9-fold to 6.71 ± 1.44 μM after oxidation compared to 0.68 ± 0.24 μM before oxidation, and was partially reverted to 1.54 ± 0.16 μM after re-reduction , to modify the free thiol groups of Cys179 and Cys203. The only native cysteine (Cys91) was mutated to serine to facilitate this analysis (see “Methods” for details). Modification of mPEG-Mal-5K adds 5 kDa per modification to the modified protein, which causes a shift of the relative mobility of the protein in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Considering that the access of mPEG-Mal-5K to Cys179 or Cys203 would likely be impeded by the nearby detergents that cover the hydrophobic exterior face of α4/α5, we expected a relatively limited efficiency for modification, and therefore this result was only used for correlative analysis. As expected, the modification percentage of xlσ1R-C179/C203 before oxidation reached merely 21.9%  and a Tyr203-to-cysteine mutant (xlσ1R-C203), and chose mPEG-Mal-5K as the blocking modifier. This reagent is bulky and has been shown to successfully modify the free thiol group of Cys179 or Cys203 in xlσ1R , we first tested the effect of mPEG-Mal-5K modification of Cys91 on PRE084 binding. The stoichiometry (number of available binding sites per protomer) and affinity (Kd) changed little for wild-type xlσ1R after mPEG-Mal-5K treatment  decreased to 0.48 ± 0.07 from 0.85 ± 0.09 for xlσ1R-C179, and to 0.69 ± 0.04 from 0.87 ± 0.01 for xlσ1R-C203  than in xlσ1R-C203 (~13%)  increased from 1.02 ± 0.05 μM to 1.95 ± 0.05 μM for the modified xlσ1R-C179, and from 0.99 ± 0.03 μM to 1.61 ± 0.09 μM for the modified xlσ1R-C203  to ensure that the folding of xlσ1R was not disrupted by the cysteine mutation(s). The xlσ1RC179/C203-S1RA structure was solved in the P21 space group, with 24 protomers in each asymmetric unit , and through hydrophobic interactions with Val81, Trp86, Met90, Tyr100, Leu102, Phe104, Tyr117, Ile121, Trp161, Ile175, Leu179, Phe181, Ala182, and Tyr203 . Since σ1R is primarily localized within cells, it has been suggested that a synthetic ligand would need to partition into and pass through, at least the plasma membrane22. Drug delivery studies also show that hydrophobic molecules do partition and enrich into the cellular membranes23. Therefore, a membrane pathway would not be a prohibitory factor for ligand entry and exit in σ1R.Third, the PATH2 access hypothesis infers that the σnt Figs. . Given t1R structures is the orientation of the transmembrane helix α1 -3-butyl)-piperidin-4-yl)malonamide (DIM-3C)21, was kindly provided by Dr. Collina and Dr. Rossino for soaking the xlσ1R crystals. Unfortunately, the highly hydrophobic DIM-3C could only be dissolved with organic solvents such as methanol and dimethyl sulfoxide, which damaged the xlσ1R crystals, and no structure was obtained for the DIM-3C soaking experiment. However, interestingly, in one of the 24 protomers of the xlσ1RC179/C203-S1RA structure, an extra electron density resembling the shape of S1RA was observed near the ligand entrance in addition to the S1RA molecule in the ligand-binding site  except 4-[2--1H-pyrazol-3-yl)oxy)ethyl]morpholine (S1RA) hydrochloride, which was purchased from Topscience Co. Ltd. .1R from Xenopus laevis  was synthesized by Genewiz  in a modified pPICZ plasmid  containing an amino-terminal tag of decahistidine and Tobacco etch virus (TEV) protease recognition site following the hemagglutinin signal peptide. Cysteine mutations were introduced by site-directed mutagenesis using QuikChange II system  according to the manufacturer’s recommendation, and all mutations were verified by sequencing. The wild-type xlσ1R and mutants were overexpressed in yeast strain GS115 (Pichia pastoris) cells by adding 1% (v/v) methanol and 2.5% (v/v) dimethyl sulfoxide (DMSO) at OD600 nm of ~1 and shaking at 20 °C for 48 h. Cell pellets were resuspended in lysis solution (LS) containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1 mM phenylmethanesulfonyl fluoride, and 2 mM β-mercaptoethanol, and lysed by an ATS AH-1500 high-pressure homogenizer  at 1300 MPa. Protein was extracted by addition of 1% (w/v) n-dodecyl-β-d-maltopyranoside  and 0.1% (w/v) cholesteryl hemisuccinate  at 4 °C for 2 h and the extraction mixture was centrifuged at 200,000 × g for 20 min at 4 °C. The supernatant was then loaded onto a cobalt metal affinity column, washed with 20 bed-volume of LS containing 3 mM DDM, 0.01% (w/v) CHS, and 20 mM imidazole pH 8.0, and eluted with LS supplemented with 3 mM DDM, 0.01% (w/v) CHS and 250 mM imidazole pH 8.0.The gene encoding the full-length wild-type σ1R was concentrated to 6-8 mg/ml and loaded onto a Superdex 200 Increase 10/300 GL column  equilibrated in 20 mM Tris-HCl pH7.5, 150 mM NaCl, 5 mM β-mercaptoethanol, 40 mM octyl-β-d-glucopyranoside (OG) and 0.001% (w/v) CHS and was further purified by size-exclusion chromatography (SEC). SEC-purified xlσ1R was then concentrated to 5–6 mg/ml as approximated by ultraviolet absorbance, and 500 nl of protein solution was mixed with an equal volume of crystallization solution manually in a vapor diffusion sitting-drop setup and was incubated at 20 °C. (1) The xlσ1Rclosed-endo crystals grew in 0.1 M NaCl, 0.1 M sodium citrate pH 5.1, 26% (v/v) PEG 400, and 10 mM KAu(CN)2. (2) The xlσ1Rclosed-PRE084 crystals grew in 0.1 M NaCl, 0.1 M sodium citrate pH 5.2, 28% (v/v) PEG 400, 10 mM KAu(CN)2, and were soaked with 0.4 mM [2-(morpholin-4-yl)ethyl] 1-phenylcyclohexane-1-carboxylate (PRE084) hydrochloride. (3) The xlσ1Rclosed-S1RA crystals grew in 0.1 M NaCl, 0.1 M sodium citrate pH 5.2, 26% (v/v) PEG 400, 10 mM KAu(CN)2, and were soaked with 0.4 mM S1RA hydrochloride. (4) The xlσ1Ropen-endo crystals grew in 0.1 M sodium HEPES pH 6.5, and 28% PEG 300. (5) The xlσ1Ropen-PRE084-co crystals grew in 0.1 M sodium HEPES pH 6.5, 28% PEG 300, and 0.4 mM PRE084 hydrochloride. (6) The xlσ1Ropen-PRE084-soak crystals grew in 0.1 M sodium HEPES pH 6.5, 28% PEG 300, and were soaked with 0.4 mM PRE084 hydrochloride. (7) The xlσ1RC179/C203-S1RA crystals grew in 0.05 M ADA pH 6.5, 24% PEG 400, 1 mM octyl-maltoside fluorinated, 0.4 mM PRE084 hydrochloride, and were soaked with 2 mM S1RA hydrochloride. The xlσ1R crystals usually appear within a week, and reach full-size in two weeks. The crystals were cryo-protected by raising the precipitant concentrations  with a 2% (v/v) incremental step, and flash-frozen in liquid nitrogen.Affinity-purified xlσ24 of the National Facility for Protein Science in Shanghai (NFPS) at the Shanghai Synchrotron Radiation Facility (SSRF). The data were indexed, integrated, and scaled using the autoPROC pipeline package 25, which includes XDS26 and AIMLESS (CCP4 package)27. All xlσ1R structures were solved by molecular replacement with Phaser28 using the published hσ1RPD144418 structure (PDB entry 5HK1) as a template. Manual model building and refinement were carried out using Coot29 and phenix.refine30, and Molprobity31 was used to monitor and improve protein geometry. The asymmetric unit of all xlσ1R structures contains 12 or 24 protomers, and so the non-crystallographic symmetry (NCS) was applied throughout the refinement to improve the map, except that for xlσ1Ropen-PRE084-co (3.10 Å) and xlσ1Ropen-PRE084-soak (2.85 Å), the NCS restraints were relaxed in the last a few rounds of refinement. The data collection and refinement statistics were generated using phenix.table_one30 and the values are listed in Table 32 with default settings.Diffraction data were collected on beamlines BL18U1 and BL19U14 stock in the xlσ1R-C179/C203 protein sample, which was SEC-purified in the absence of any reducing reagent, to a final concentration of 200 μM for 1 h at the room temperature. The oxidation reaction was stopped by the addition of 10 mM EDTA pH 8.0 for 10 min at room temperature, and the protein sample was re-purified by SEC. For the re-reduction experiment, the oxidized xlσ1R-C179/C203 sample was incubated with 60 mM β-mercaptoethanol at 4 °C for 12 h, and the protein sample was then re-purified by SEC. The oxidized or re-reduced xlσ1R-C179/C203 samples were subjected to ITC or SDS-PAGE analysis.The disulfide bond formation between Cys179 and Cys203 was catalyzed by adding a freshly prepared CuSO1R was performed with a bulky thiol-modifying reagent, methoxypolyethylene glycol maleimide 5000 33. Briefly, the SEC-purified xlσ1R protein in the absence of β-mercaptoethanol were incubated with 1 mM mPEG-Mal-5K at room temperature for 1 h, and the modification reaction was stopped by addition of β-mercaptoethanol to a final concentration of 10 mM before another SEC purification to remove any free mPEG-Mal-5K. For the entrance blocking experiment, the modified xlσ1R-C179 or xlσ1R-C203 proteins were subjected to ITC or SDS-PAGE analysis. For the assessment of disulfide formation in xlσ1R-C179/C203, the modified sample was analyzed by SDS-PAGE. To reduce background modification and facilitate gel analysis, the only native cysteine (Cys91) was mutated to serine in all samples for the disulfide analysis.Modification of wild-type and cysteine mutants of xlσ34. Briefly, the gel image was first converted to grayscale, and equal-sized rectangles were drawn to encompass the bands of interest to measure their intensities. The intensity of a background rectangle was also measured for background correction. The modification ratio of the xlσ1R variants was calculated by the following equation: modification ratio (%) = (modified band intensity)/(modified + unmodified band intensities) × 100.Protein bands in SDS-PAGE gels were visualized by Coomassie blue staining, and the band intensities were quantified using the ImageJ software1R was performed using a MicroCal iTC200 microcalorimeter . The xlσ1R samples and PRE084 were prepared in the titration buffer containing 20 mM sodium HEPES pH 7.5, 150 mM NaCl, 1 mM DDM, and 0.005% (w/v) CHS. The first injection was 0.2 µl in volume and the subsequent injections were 2.0 µl. Before the data collection, the system was equilibrated to 25 °C with the stirring speed set to 750 rounds per min. The titration curve of PRE084 was generated with nineteen 2.0-µl injections at 90-s intervals. All the xlσ1R titration data were best fitted by a one-site binding model using ORIGIN 7, and calculations were performed using Microsoft Excel for Mac 2016. The values of the available binding site number per protomer (stoichiometry) and the equilibrium dissociation constant (Kd) were determined from the average of three to six biologically independent ITC measurements and were expressed as mean ± SD in the text. Two-tailed Student’s t-test was performed for statistical analysis, and details were described in relevant figure legends.The ITC measurement for the binding of PRE084 to xlσFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary