An asymmetric constitutional isomer of the thermally activated delayed fluorescence (TADF) host DPEPO, named 24′DPEPO, endowed deep-blue TADF diodes with state-of-the-art performance, including external quantum efficiency beyond 20%. DPEPO is the most popular high-energy-gap blue TADF host with steric ortho-substituted diphenylphosphine oxide (DPPO) groups for intermolecular interaction suppression, but suffers from serious efficiency roll-off due to its weak electroactivity. On the contrary, para-substituted DPPO with small steric hindrance is superior in intramolecular electronic coupling. In this work, four constitutional isomers of DPEPO are constructed as diphenylether (DPE) with two diphenylphosphine oxide (DPPO) groups substituted at either the 2 or 4 position, namely 22′DPEPO (viz.DPEPO), 24DPEPO, 24′DPEPO and 44′DPEPO, respectively. On the basis of separation configuration, the steric effect and electroactivity of ortho- and para-substituted DPPOs are successfully integrated in 24′DPEPO, accompanied by remarkably reduced intermolecular interactions due to its unsymmetrical configuration. Compared to its congeners, 24′DPEPO has a rigid structure and locally excited states similar to 22′DPEPO for interaction suppression and improved charge mobility comparable to 44′DPEPO for charge flux balance. Significantly, by virtue of the predominant orientation effect of ortho-DPPO on the T1 location, its T1 state is extremely condensed onto a single phenyl, protected from intermolecular interactions by its remaining five phenyls at its maximum extent. Consequently, 24′DPEPO endowed its DMAC-DPS-based deep-blue devices with state-of-the-art performance, including high color purity with chromaticity coordinates of , external quantum efficiency (EQE) beyond 20% and EQE roll-off as low as 32% at 1000 cd m–2. It is shown that the device performance of 24′DPEPO was far beyond simple integration of those of 22′DPEPO and 44′DPEPO, verifying the significance of host optimization.The similarity of thermally activated delayed fluorescence (TADF) dyes and their hosts as pure organic molecules makes hosts predominant in intermolecular interactions and crucial to exciton harvesting and utilization in TADF diodes.  Meanwhiion (IC) .12 In coensation ; (ii) reDPEPO is the most popular host for blue TADF diodes. PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdwLlLl" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O groups give rise to its high T1 value of ∼3.0 eV for efficient energy transfer to blue TADF dyes, e.g. bis[4-phenyl]sulfone (DMAC-DPS) (T1 = 2.90 eV)DMAC-DPS as the emitter reached ∼20%, however, this was accompanied by remarkable roll-off of more than 90% at 1000 cd m–2, indicating serious exciton quenching.DPEPO may provide a feasible way to clarify the key determinants of EL performance for deep blue TADF host materials.It is known that the CT-type exciton is labile and highly sensitive to the environment.DPEPO with the collective name of mDPEPO are constructed as bis(diphenylphosphoryl) diphenylether with two diphenylphosphine oxide (DPPO) groups at either the 2 or 4 position of their diphenylether (DPE) cores, named 22′DPEPO (viz.DPEPO), 24DPEPO, 24′DPEPO and 44′DPEPO, respectively , an EQE beyond 20% and EQE roll-off as low as 32% at 1000 cd m–2. The efficiencies of 24′DPEPO-based devices were improved by 20% in comparison to those employing 22′DPEPO as the host; meanwhile, the roll-offs of the former were halved. It is clear that host engineering is a gateway to resolving the key issues of TADF devices in exciton harvesting and utilization.In this contribution, four constitutional isomers of ectively . DPPO at2.2.1.ortho-positions of 22′DPEPO are superior in their steric effect; while, DPPOs in 44′DPEPO can enhance intramolecular coupling with an electron-donating ether bridge at the para-position  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O group on electron cloud distribution.1 state would be related to its substitution position. Therefore, with asymmetrical DPPO substitutions, the T1 states of the molecules can be condensed into a designated location by DPPO with a dominant orientation effect. The narrow T1 location can amplify the protective action of surrounding groups and further reduce its probability of involvement in intermolecular interactions. Regarding DPEPO-type analogues, since all of their phenyls are separated by insulating linkages, it can be expected to confine their T1 states into a single phenyl, which is almost the minimum for conjugated compounds with respect to spatial scale.More significantly, in our recent works, it was shown that DPPO substitution can regulate T24DPEPO and 24′DPEPO, incorporating both ortho- and para-linked DPPO groups were designed. Two DPPOs in 24DPEPO are bonded with the same phenyl, while those in 24′DPEPO are respectively bonded with two different phenyls of DPE. The superposition of two DPPOs in 24DPEPO may render the mutual acceleration or counteraction of their effects. On the contrary, by virtue of the insulating ether bridge, the two separated DPPOs of 24′DPEPO can perform their functions independently. In consequence, it is most likely for 24′DPEPO to integrate the merits of 22′DPEPO and 44′DPEPO. Simultaneously, the multi-insulating linkage establishes the similarity of mDPEPO to the maximum extent, excluding the interference from undesirable intramolecular interplay and conjugation variation, which simplifies the influencing factors of their device performance into electrical and excited-state characteristics.With these considerations, two other asymmetrical isomers, namely mDPEPO can be conveniently prepared through Pd-catalyzed phosphorylation with good yields of more than 50%. Their chemical structures were fully characterized on the basis of NMR spectra, mass spectra and elementary analysis. Among mDPEPO, the biggest steric hindrance in 22′DPEPO increases its intramolecular tension, rendering its lowest temperature of decomposition (Td) as 322 °C; while its rigid and monosymmetrical structure results in its highest melting point (Tm) of 280 °C , taking into account computational accuracy and cost.For insight into the nature of the effects of DPPO substitution on electrical and excited-state characteristics of mDPEPO at ground (S0), the first singlet (S1) and T1 states are shown in ortho-substituted DPPOs, the dihedral angle of DPE in 22′DPEPO at the S0 state is the largest among mDPEPO, which is almost preserved at its S1 and T1 states. The dihedral angles of DPE in 44′DPEPO at these states are the smallest, in accord with the smaller steric hindrance of its DPPOs at the para-position. Compared to the ground state, its excited-state configurations are adjusted. Nevertheless, 24DPEPO shows the largest configuration variation at excited states, which is ascribed to the rotational phenyl of its DPE. In this sense, both ortho- and para-DPPO substitution can reduce excited-state structural relaxation. Consequently, the molecular configurations of 24′DPEPO at S0, S1 and T1 states are almost unchanged, accompanied by the dihedral angles of DPE close to those of 22′DPEPO. In comparison to 24DPEPO, the fixed molecular configurations of 24′DPEPO and 22′DPEPO make them superior in reducing relaxation-induced excited-energy loss, which is beneficial to improving luminescent efficiency.The optimized molecular configurations of mDPEPO reveal the influence of DPPO substitution position on hole and electron injecting ability, respectively. Although the frontier molecular orbitals (FMO) of 22′DPEPO are respectively localized on its DPE and DPPO groups, its LUMO energy level is the highest among mDPEPO, indicating the weakest electron-withdrawing ability of its DPPOs at the 2-position  for position . The sit1 and T1 energies of mDPEPO are almost the same as ∼4.7 and ∼3.5 eV, supporting positive energy transfer to DMAC-DPS with the S1 and T1 value of ∼2.9 eV  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O groups. Interestingly, 24′DPEPO shows a T1 state that is thoroughly localized on the 2-DPPO substituted phenyl, reflecting the dominant orientation effect of ortho-substituted DPPO on the T1 state. The T1 state of 24′DPEPO is extremely condensed on a single phenyl as one of the smallest conjugated units, which is embedded by its remaining five phenyls. Compared to its isomers, the exposure degree of the T1 state of 24′DPEPO is doubtlessly the smallest, which can support the most effective suppression of collision-induced quenching effects.The calculated S ∼2.9 eV . The simmDPEPO was further evaluated with natural transition orbitals (NTO) of the first singlet and triplet excitations  state transition; while, for 24DPEPO, “holes” are concentrated to one phenyl of DPE, and for 44′DPEPO, “holes” are partially dispersed on DPPO groups, characteristic of hybridized local and charge transfer excited states. Nevertheless, the biggest oscillator strength (f) suggests the LE transition of DPE as the major part for 44′DPEPO, while CT transition between two phenyls of DPE is dominant for 24DPEPO with the smallest f. NTOs of S0 → T1 states for mDPEPO reveal an LE character with overlapped “hole” and “particle” locations. In accord with the DFT results, triplet states of 22′DPEPO and 44′DPEPO are dispersed on their DPE, while only a single phenyl of 24DPEPO and 24′DPEPO is involved in their triplet transitions. Regarding CT character of excitons on TADF dyes, LE-dominant excited states of hosts can provide uniform and apparently neutral matrices to restrain Coulomb interaction induced CT exciton dissociation.The nature of electronic transitions for the excited states of itations  and S4†.24′DPEPO successfully integrates the merits of ortho- and para-DPPO substitutions, including excited structural stability, singlet characteristics comparable to 22′DPEPO and FMO energy levels close to 44′DPEPO. In addition to significantly reinforced T1 state protection through an extremely condensed T1 location on the minimum unit phenyl, 24′DPEPO shows superiority in quenching suppression and charge balance. In contrast, 24DPEPO exhibits some shortages of serious excited structural relaxation and a CT-dominant S1 characteristic.It is shown that 2.3.mDPEPO in dilute solutions (10–6 M in CH2Cl2) consist of three bands at around 230, 270 and 300 nm, corresponding to π → π* transitions of DPPO and DPE and the n → π* transition of DPE, respectively  are exactly the same in range and profile. Meanwhile, the FL spectrum of 44′DPEPO reveals a blue shift of ∼10 nm. Nonetheless, there are large-range spectral overlaps between FL emissions of mDPEPO and absorption of DMAC-DPS from 275 to 350 nm, facilitating Förster resonance energy transfer (FRET). The low-temperature time-resolved phosphorescence (PH) spectra of mDPEPO are identical, with the same peaks and profiles, which is in accord with their similar T1 transition characteristics as shown by TDDFT simulation  spectra of inset of . EstimatmDPEPO in the solid state were further investigated to exclude solvent effects  are also comparable to ∼7 μs  of only 59%  spectra of vacuum-evaporated PO films . The emionly 59% . Further24′DPEPO and 22′DPEPO are identical, originating from their similar excited-state characteristics as TDDFT simulated.In consequence, the optical properties of 2.4.mDPEPO consist of two irreversible peaks characteristic of DPE and DPPO, respectively. Estimated with onset voltages of the anodic peaks, the HOMO energy level of 24′DPEPO is equivalent to that of 44′DPEPO at –6.65 eV, which is 0.1 eV lower than those of 22′DPEPO and 24DPEPO  analysis . The oxi 24DPEPO . On the 3 (6 nm)|mDPEPO (100 nm)|MoO3 (6 nm)|Al for hole only and ITO|LiF (1 nm)|mDPEPO (100 nm)|LiF (1 nm)|Al for electron only, where MoO3 and LiF respectively served as hole- and electron-injecting layers, were fabricated to evaluate the intrinsic carrier transporting ability of mDPEPO (J) remarkably larger than hole-only J. According to field-dependent space charge limited current model, the hole mobility (μh) of 44′DPEPO is estimated to be 1.20 × 10–7 cm2 V–1 s–1, which is 10 fold that of 24′DPEPO and 100 fold those of 22′DPEPO and 24DPEPO  is the lowest at 7.72 × 10–9 cm2 V–1 s–1, which is only one tenth of that of 22′DPEPO. In contrast, the μe of 24′DPEPO at 4.02 × 10–6 cm2 V–1 s–1 is dramatically improved by 3 orders of magnitude, which is almost equivalent to that of 44′DPEPO. According to the DFT results, both DPE and DPPO are involved in unoccupied molecular orbitals and incorporated into electron transportation |NPB (70 nm)|mCP (5 nm)|mDPEPO:DMAC-DPS |mDPEPO (5 nm)|Bphen (30 nm)|LiF (1 nm)|Al, in which MoO3 and LiF served as the hole and electron-injecting layer, NPB and Bphen are 4,4′-bis[N-(1-naphthyl)-N-phenylamino]biphenyl and 4,7-diphenyl-1,10-phenanthroline as hole and electron transporting layers and mCP  and mDPEPO were used as exciton-blocking layers, respectively  coordinates of , owing to their rigid structures and LE-featured excited states  and , respectively. The remarkably increased intensity of the long-wavelength part in the EL emission from 24DPEPO-based devices was actually due to the decreased intensity of the short-wavelength part, which is readily quenched through nonradiative transitions during host–dopant interactions.All of the devices showed deep blue emissions peaking at 460 nm at 1000 cd minset in . In compd states . In contI–V characteristics of these light-emitting devices showed a trend consistent with the charge mobility of their host materials , 21.5 lm W–1 for power efficiency (PE) and 16.7% for EQE, which were about 20% higher than those of 44′DPEPO-based devices -4,5-dicyanobenzene (2CzPN) employing 24′DPEPO also showed higher efficiencies and reduced roll-off in contrast to 22′DPEPO, displaying the universality of the host design strategy for 24′DPEPO  as an internal standard. Molecular masses were determined using a FINNIGAN LCQ Electro-Spraying Ionization-Mass Spectrometer (ESI-MS), or a MALDI-TOF-MS. Elemental analyses were performed on a Vario EL III elemental analyzer. Absorption and photoluminescence (PL) emission spectra of the target compounds were measured using a SHIMADZU UV-3150 spectrophotometer and a SHIMADZU RF-5301PC spectrophotometer, respectively. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed on Shimadzu DSC-60A and DTG-60A thermal analyzers under a nitrogen atmosphere at a heating rate of 10 °C min–1. Cyclic voltammetric (CV) studies were conducted using an Eco Chemie B. V. AUTOLAB potentiostat in a typical three-electrode cell with a platinum sheet working electrode, a platinum wire counter electrode, and a silver/silver nitrate (Ag/Ag+) reference electrode. All electrochemical experiments were carried out under a nitrogen atmosphere at room temperature in dichloromethane. Phosphorescence spectra were measured in dichloromethane using an Edinburgh FPLS 920 fluorescence spectrophotometer at 77 K cooled by liquid nitrogen with a delay of 300 μs using the Time-Correlated Single Photon Counting (TCSPC) method with a microsecond pulsed xenon light source for 10 μs to 10 s lifetime measurement, and a synchronization photomultiplier for signal collection and the Multi-Channel Scaling Mode of a PCS900 fast counter PC plug-in card for data processing. The absolute PLQY of the films was measured with an integrating sphere.2, bromide (1 mmol), NaAc (1.1 mmol), Pd(Ac)2 (0.05 mmol) and Ph2PH (1.1 mmol) were dissolved in DMF (10 mL) and heated to reflux for 24 h. After cooling to room temperature, water (10 mL) was added to the system, which was then extracted with CH2Cl2 (3 × 3 mL). The organic layer was combined and dried with anhydrous sodium sulfate. The solvent was removed in vacuo to afford the crude phosphine intermediate product. Then, the phosphine was dissolved in CH2Cl2 (10 mL). H2O2  was added to the solution dropwise and stirred for 2 h at 0 °C. Then, the mixture was extracted using CH2Cl2 (3 × 3 mL). The organic layer was combined and dried with anhydrous sodium sulfate. The solvent was removed in vacuo, and then the residue was purified by flash column chromatography to afford the phosphine oxide product : δ = 8.009–7.957 , 7.840–7.775 , 7.717–7.606 , 7.566–7.284 , 6.862–6.821 , 6.572–6.550 ppm ; LDI-TOF: m/z (%): 570 (100) [M+]; elemental analysis (%) for C36H28O3P2: C 75.78, H 4.95; found: C 75.82, H 4.97.393 mg of white powder with a yield of 69%. 1H NMR : δ = 8.062–8.007 , 7.767–7.719 , 7.667–7.616 , 7.589–7.534 , 7.506–7.315 , 6.930–6.897 , 6.665–6.638 ppm ; LDI-TOF: m/z (%): 570 (100) [M+]; elemental analysis (%) for C36H28O3P2: C 75.78, H 4.95; found: C 75.80, H 4.95.439 mg of white powder with a yield of 77%. 1H NMR : δ = 7.691–7.621 , 7.563–7.523 , 7.482–7.438 , 7.105–7.078 ; LDI-TOF: m/z (%): 570 (100) [M+]; elemental analysis (%) for C36H28O3P2: C 75.78, H 4.95; found: C 75.77, H 4.94.428 mg of white powder with a yield of 75%. 3.2.DFT computations were carried out with different parameters for structure optimizations and vibration analyses. The ground states and triplet states of molecules in vacuum were optimized by the restricted and unrestricted formalism of Beck's three-parameter hybrid exchange functional293.3.–1 onto the ITO substrate sequentially at a pressure below 4 × 10–4 Pa. Onto the electron-transporting layer, a layer of LiF with 1 nm thickness was deposited at a rate of 0.1 nm s–1 to improve electron injection. Finally, a 100 nm-thick layer of Al was deposited at a rate of 0.6 nm s–1 as the cathode. The emission area of the devices was 0.09 cm2 as determined by the overlap area of the anode and the cathode. After fabrication, the devices were immediately transferred to a glove box for encapsulation with glass cover slips using epoxy glue. The EL spectra and CIE coordinates were measured using a PR650 spectra colorimeter. The current–density–voltage and brightness–voltage curves of the devices were measured using a Keithley 4200 source meter and a calibrated silicon photodiode. All the measurements were carried out at room temperature under ambient conditions. For each structure, five devices were fabricated to confirm the performance repeatability. To make conclusions reliable, the data reported herein were the closest to the average results.Before loading into a deposition chamber, the ITO substrate was cleaned with detergents and deionized water, dried in an oven at 120 °C for 4 h, and treated with oxygen plasma for 3 min. Devices were fabricated by evaporating the organic layers at a rate of 0.1–0.2 nm s4.DPEPO-type host materials named mDPEPO as constitutional isomers with two DPPO groups substituted on a DPE core at either the ortho- or para-position were designed and prepared. The steric effect of ortho-substituted DPPO and the electron coupling effect of para-substituted DPPO were successfully integrated through the separation configuration of 24′DPEPO, giving rise to its harmonious excited-state characteristics and electrical performance inherited from 22′DPEPO and 44′DPEPO, respectively. The asymmetrical configuration and the dominant orientation effect of ortho-substituted DPPO on the T1 location extremely condense the T1 state of 24′DPEPO onto a single phenyl, completely protected from intermolecular interactions. Consequently, 24′DPEPO endowed its DMAC-DPS-based deep blue TADF diodes with state-of-the-art performance featuring high EQE beyond 20% and low roll-off at 32% at 1000 cd m–2, which was dramatically improved in comparison to those of 22′DPEPO and 44′DPEPO and successfully demonstrated its potential. This showed that besides common energy level optimization, more delicate and purposeful modulation on the optoelectronic properties of host materials is crucial for developing high-performance TADF diodes.A series of Supplementary informationClick here for additional data file.


At the molecular level this methylation could alter local RNA structure, and/or modulate the binding of specific proteins. We have previously shown that trans-Hoogsteen-sugar (sheared) A:G base pairs can be completely disrupted by methylation, and that this occurs in a sub-set ofD/D k-turn structures. In this work we have investigated to what extent sequence context affects the severity with which inclusion of N6-methyladenine into different A:G base pairs of a standard k-turn affects RNA folding and L7Ae protein binding. We find that local sequence has a major influence, ranging from complete absence of folding and protein binding to a relatively mild effect. We have determined the crystal structure of one of these species both free and protein-bound, showing the environment of the methyl group and the way the modification is accommodated into the k-turn structure.N Inclusi6-methyladenine at the A1n position resulted in no folding being observed up to a concentration of 0.1 M Mg2+ , and undergo folding in response to binding. AfL7Ae binding was studied using gel electrophoresis  and 0.67 (A3b)  . A greatfor Kt-7 , with th6-methyladenine 2b Kt-7 underwent folding on addition of Mg2+ ions, we investigated the structure using X-ray crystallography. Crystals of self-complementary RNA containing two Kt-7 k-turns were obtained with and without bound AfL7Ae, at resolutions of 1.99 and 2.09 Å respectively. The structure of the k-turn containing N6-methyladenine 2b is closely similar in the L7Ae bound and protein free states, superimposing with an all-atom RMSD of 0.52 Å . The structure of the complex is shown in AfL7Ae bound to unmodified Kt-7  is the prevailing Mg2+ ion concentration, AK is the apparent association constant for metal ion binding and n is a Hill coefficient. Data were fitted to this equation by nonlinear regression. The metal ion concentration at which the transition is half complete is given by [Mg2+]1/2 = (1/AK)n1/.where AfL7Ae protein was titrated in to the RNA and added to a final concentration of 2 µM. EFRET as a function of AfL7Ae concentration was fitted to:The same hybrid RNA species were used to observe L7Ae binding-induced folding, and FRET efficiency was calculated using the same approach. 0E is the initial FRET efficiency in the absence of added protein, FRET∆E is the full range of the change in FRET efficiency, AK is the apparent association constant and TP and TR are the total concentration of AfL7Ae and RNA respectively.where AfL7Ae protein to the Kt-7 k-turn RNA and its modified versions was analysed by native gel electrophoresis. 200 nM Cy3-labeled RNA (the same species as used in the FRET experiments) was incubated in 45 mM Tris-borate (pH 8.3) with various concentrations of the AfL7Ae protein in a final volume of 10 µl at 7°C for at least 1 h. An equal volume of loading buffer containing 10% glycerol and 45 mM Tris-borate (pH 8.3) was added, and the samples were electrophoresed in 10% native polyacrylamide gels in 45 mM Tris-borate (pH 8.3) at 20 V/cm at 7°C for 1 h. Fluorescent RNA was visualized using a Typhoon 9500 fluorimager .Binding of 6-methyladenine). This sequence is self-complementary and forms two Kt-7 k-turns with two-fold symmetry. A solution of 1 mM RNA in 5 mM Tris.HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2 was incubated for 1 min at 95°C. Crystals were grown by vapor diffusion using drops prepared by mixing 1 μL of the RNA with 1 μL of a reservoir solution comprising 0.1 M KCl, 0.002 M spermine tetrahydrochloride, 0.05 M Bis-Tris (pH 8.0) and 15% w/v PEG 2000 MME at 17°C. Crystals appeared after 10 days. They were transferred into the reservoir solution with 30% PEG200 for ~ 3 s. The crystals were flash frozen by mounting in nylon loops and plunging into liquid nitrogen.A 19 nt RNA of sequence GGCGAAG(6mA)ACCGGGGAGCC was crystalized , 100 mM NaCl, 10 mM MgCl2 was incubated for 1 min at 85°C followed by slow cooling. Crystals were grown by vapor diffusion using drops prepared by mixing 1.0 μL of the RNA–protein complex with 1 μL of a reservoir solution comprising 0.01 M spermine tetrahydrochloride, 0.05 M MES (pH 6.5), 25% v/v PEG 400 at 17°C. Crystals appeared after 5 days.A self-complementary 21 nt RNA oligonucleotide CGGCGAAG(6mA)ACCGGGGAGCCG was crystalized as a complex bound to All the crystals were characterized in-house with a MicroMax HF007 copper rotating anode X-ray generator equipped with an ACTOR sample changer system and a Saturn 944HG+ CCD detector (Rigaku). Suitable crystals were stored and subsequently used to measure full datasets on beamline I03 of Diamond Light Source (Harwell, UK. Data were processed by XIA2 .AfL7Ae protein were determined by molecular replacement with Phenix Phaser. The k-turn search model was taken from PDB 4CS1, and the k-turn AfL7Ae complex model was taken from PDB 4BW0. The resulting electron density maps revealed the remaining RNA density, and the model was built de novo on the basis of the difference map. Structural models were built in Coot [The structures of the HmKt-7 variant A2bm6A without and with  in Coot  and refi in Coot . Omit ma in Coot  and the


Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙+ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from  However, the pharmacological activities (in vitro and in vivo) of pterostilbene are usually better than those of resveratrol + ion of the pterostilbene monoglucoside. For compound 3, the main signal in the mass spectrum in negative mode was at m/z 579.21 belonging to the M − [H]+ ion of the pterostilbene diglucoside.Peaks 2 and 3 were purified by semipreparative HPLC on a C-18 column as described. The molecular weight was determined by MS using electrospray and QTOF analyzer see . For comThe other peaks in the chromatogram of The progress of pterostilbene glucosylation under the assayed conditions was further studied . After 13J coupling between the protons H1 and H2 of the glucose moiety, which is 3.6 Hz. 1H- and 13C-NMR chemical shifts δ for position 1- of Glc also supported this configuration . The β-glucosylated derivative has been described  and (Saccharomyces cerevisiae and cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme 3.0L) were kindly provided by Novozymes A/S . Toruzyme 3.0L was partially purified by a PD-10 desalting column . CGTase from Bacillus macerans (cyclodextrin glucanotransferase) and α-glucosidase from Aspergillus niger (Transglucosidase L) were kindly supplied by Amano Enzyme Inc. . The β-galactosidase from Bacillus circulans (Biolactase NTL Conc. 2x) was provided from Biocon . The β-fructofuranosidase from Xanthophyllomyces dendrorhous was obtained as previously described [The β-fructofuranosidase from escribed .S. cerevisiae (var. diastaticus) (STA1), a 2.35 kb EcoRI/SacII DNA fragment containing the STA1 gene from S. cerevisiae (var. diastaticus) [Pichia pastoris expression vector pPICZalpha. The resulting plasmid was used to transform P. pastoris strain X-33 according to the manual for protein expression in Pichia . To obtain STA1 protein expressed in P. pastoris, transformants were grown, and the heterologous protein was produced and purified as described in a previous work [For the production of recombinant amyloglucosidase from taticus)  was clonous work . Briefly2O (0.7 mL) and DMSO (0.2 mL). Partially purified CGTase from Thermoanaerobacter sp. (0.1 mL) was then added to a final concentration of 10% (v/v). The mixture was kept at 60 °C in an orbital shaker  at 150 rpm. Aliquots (150 μL) were withdrawn at intervals, passed through 0.45 μm nylon filters (Cosela), diluted with 150 μL acetonitrile to stop the reaction and analyzed by HPLC. For the hydrolysis of polyglucosylated derivatives, the reaction with CGTase was first carried out under the above conditions during 10 h. Then, it was cooled and centrifuged 15 min at 7800× g to remove non-solubilized starch. The supernatant was collected and amyloglucosidase from S. diastaticus (STA1)  was then added and the mixture kept at 40 °C. Aliquots of 150 µL were taken during 3 h, diluted with 150 µL of acetonitrile and analyzed by HPLC.Pterostilbene (5 mg) and partially hydrolyzed starch (100 mg) were dissolved in a mixture of Hv/v) at 0.8 mL/min during 10 min. Both solvents contained 0.1% (v/v) of formic acid. The analysis was also performed with a Zorbax Eclipse Plus C18 column . The starting mobile phase was H2O/acetonitrile 85:15 (v/v), and a gradient to H2O/acetonitrile 5:95 (v/v) was performed in 12 min. This mobile phase was maintained for 2 min and the column was further equilibrated to the initial conditions. The flow rate was 0.8 mL/min, and the column temperature was kept constant at 40 °C.HPLC analysis was performed using a quaternary pump  coupled to an autosampler . The column was a Kromasil-NH2 . The injection volume was 20 µL. The temperature of the column was kept constant at 40 °C. The detection of peaks was carried out using a photodiode array detector  and peaks were analyzed using the Varian Star LC workstation 6.41. The mobile phase was acetonitrile/water 80:20 (2O/acetonitrile 85:15 (v/v), and a gradient to H2O/acetonitrile 5:95 (v/v) was performed in 12 min. Both solvents contained 0.1% (v/v) of formic acid. The flow rate was 7 mL/min, and the column temperature was kept constant at 40 °C. After collecting the glucosylated products, the mobile phase was eliminated by rotary evaporation in an R-210 rotavapor . The isolated products were characterized by mass spectroscopy and NMR.The glucosylation of pterostilbene was scaled up. The reaction mixture contained pterostilbene (50.7 mg), soluble starch (1 g), partially purified Toruzyme 3.0 L (1 mL), 7 mL of water and 2 mL of DMSO. The mixture was incubated for 16 h at 60 °C with orbital shaking  at 150 rpm. The mixture was then cooled and concentrated, and the glucosylated derivatives of pterostilbene were purified using semipreparative HPLC. The column was a Zorbax Eclipse XDBC-18 . A three-way flow splitter at 1/10  was employed. The starting mobile phase was H4OH as ionizing phase) in negative reflector mode.The molecular weight of purified pterostilbene derivatives was assessed using a mass spectrometer with hybrid QTOF analyzer . Samples were analyzed by direct infusion and ionized by electrospray (with methanol containing 1% of NHd6 (ca. 7 mM), were recorded on a IVDr 600 spectrometer  equipped with a BBI probe with gradients in the Z axis, at a temperature of 300 K. Chemical shifts were expressed in parts per million (ppm). Residual DMSO-d5 signal was used as internal reference (2.5 ppm). All the employed pulse sequences were provided by Bruker. For the DEPT-HSQC experiment, values of 8 ppm and 1 K points, for the 1H dimension, and 165 ppm and 256 points for the 13C dimension, were used. For the homonuclear COSY and NOESY experiments, 8 ppm windows were used with a 1 K × 256 point matrix. For the NOESY the mixing time was 500 ms.The structure of the monoglucosylated derivative was determined using a combination of 1D and 2D  standard NMR techniques. The spectra of the sample, dissolved in DMSO-+ cation was generated from ABTS solution (7 mM) with potassium persulfate (2.45 mM) for 15 h. The radical cation absorbed at 734 nm and was stable for two days. ABTS˙+ was diluted in ethanol to 0.7 ± 0.02 absorbance units at 734 nm. The antioxidant solution  was added to 230 µL of adjusted ABTS˙+ solution. The decrease of absorbance of ABTS˙+ solution was monitored at 734 nm during 6 min using a microplate reader . The decrease of absorbance was determined measuring the area under the curve. The reference antioxidant was (R)-Trolox. The TEAC values were expressed as the concentration (µM) at which the compound decreases the same absorbance as 1 µM (R)-Trolox.The ABTS˙A saturated solution of pterostilbene or its monoglucoside was prepared in water at 30 °C with stirring for 24 h. Then, the solution was filtered, conveniently diluted with water and analyzed by HPLC as previously described.SH-S5Y5 neurons were cultured in collagen-pretreated petri-dishes with DMEM-F12 medium supplemented with penicillin/streptomycin and 10% inactivated fetal bovine serum (iFBS). RAW 264.7 macrophages and HT-29 colon cancer cells were cultured in DMEM high glucose medium supplemented with penicillin/streptomycin and 10% iFBS. MRC5 were cultured in DMEM low glucose medium supplemented with glutamine, penicillin/streptomycin and 10% iFBS.4 neurons per well in a 100 µL volume and with 24 h of incubation before the compound addition. Macrophage assays were done in 96 well plates by seeding 2.5 × 104 macrophages per well in a 100 µL volume with 4 h of incubation before the compound addition. MRC5 and HT-29 assays were done in 96 well plates by seeding 5 × 104 cells per well in a 100 µL volume and with 24 h of incubation before the compound addition. Tested compounds dissolved in DMSO were then added at different final concentrations  to determine compound toxicity. Final DMSO percentage in each cell was adjusted to 1%. Cell viability was evaluated during 24 h (SH-SY5Y and RAW 264.7 cells) or 48 h (MRC5 and HT-29 cells) after the addition of the compounds, following the mitochondrial MTT assay, according to manufacturer. Averages and standard deviations of at least eight different readings from various experiments were calculated.Neuron assays were done in collagen-pretreated 96 well plates by seeding 2 × 102O:DMSO 80:20 as solvent, 60 °C) using partially hydrolyzed starch as glucosyl donor and CGTase as biocatalyst. Although the antioxidant activity decreased upon glucosylation, the monoglucoside presented higher solubility in water than the aglycon, and it was also less toxic for several cell lines. The synthesized derivative could be of interest in the nutraceutical, cosmetic and biomedical industries, as occurs with other glucosides of polyphenols obtained by biocatalytic routes [We have synthesized a novel α-glucoside of pterostilbene under mild conditions (Hc routes . However


Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 < 5.0 μg/mL). Among the compounds isolated from these extracts, laevigatol B (1) from Lobophytum crassum and L. laevigatum, (24S)-ergost-4-ene-3-one (2) from Sinularia dissecta, astropectenol A (3) from Astropecten polyacanthus, and cholest-8-ene-3β,5α,6β,7α-tetraol (4) from Diadema savignyi showed inhibitory activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM). Laevigatol B (1) and 5α-cholest-8(14)-ene-3β,7α-diol (5) exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against  Trypanosoma brucei subspecies), Chagas’ disease (caused by T. cruzi), and leishmaniasis , which affect many millions of people worldwide, although the number of new infections currently appears to be decreasing [Neglected tropical diseases (NTDs) are life threatening or disabling infections affecting more than a billion people worldwide. People suffering from NTDs hence constitute an unattractive market to private sector research and development investment . This sicreasing . This decreasing  and may creasing ,8.Marine environments, particularly in the tropics, have greater species richness than forests. These diverse species are capable of producing a wide variety of the chemical compounds with unique structures and functions, and many of these compounds can be used for the development of novel drugs . Thus, rT. brucei and T. cruzi (EC50 < 5.0 μg/mL) with no toxicity on the mammalian HEK293T (human embryonic kidney) and HepG2 (human hepatoma) cell lines. We herein report on the anti-trypanosomal activity of the major pure constituents from five of the most active extracts against the mentioned Trypanosoma species. The respective inhibitory effects as well as their potential cytotoxicity were investigated. Our results show that these compounds 1–5 . The library was composed of both fractionated single components as well as sub-fractionated plant and marine extracts (see supporting data). The screening was performed at a single concentration of 20.0 μM for purified natural products and sub-fractionated extracts were tested at 50.0 and 100 μg/mL. Biological activity data were normalized using the growth inhibition of pentamidine (positive control) at 120 nM and that of DMSO 0.5% (negative control) as the maximum and minimum values. The Z’ factor for the controls was calculated to be 0.83 using the equation defined as:C+/σC− are the standard deviation (SD) of the positive/negative controls and μC+/μC− are the mean values, indicating that the assay was performed with an excellent window between the two controls  and were therefore selected for a more detailed study of the activity of their chemical constituents.To compare the activity of our natural products to the control, the dose responsive 1) from Lobophytum crassum and L. laevigatum [S)-ergost-4-ene-3-one (2) from Sinularia dissecta [3) and 5α-cholest-8(14)-ene-3β,7α-diol (5) from Astropecten polyacanthus [β,5α,6β,7α-tetraol (4) from Diadema savignyi [T. brucei as well as their cytotoxicity against human cell lines (HEK293T and HepG2) were determined. The data are reported in The major constituents of the selected methanolic extracts with high activity were previously isolated in our laboratory (Y.H.K.). These compounds were laevigatol B -edissecta , astropeacanthus  and cholsavignyi , respect1‒4 displayed activity against T. brucei and no significant cytotoxicity against either tested cell line up to a concentration of 100 µM (the highest concentration tested). Only compound 5 did not show any significant anti-trypanosomal activity up to 30.0 µM. The anti-trypanosomal effect of compounds 1‒4 showed a clear dose-dependency  being the most active with an EC50 value of 1.57 ± 0.14 µM. Although its activity was about 100 times lower than that of the positive control (pentamidine) with an EC50 of only 0.015 ± 0.003 µM, it is worth noting that the selectivity index (SI) of 3 was markedly higher (63.7 vs. 26.7), so that this compound certainly represents an interesting starting point for further optimization. The relatively high SI value of compound 3 indicates that in this case anti-trypanosomal activity is based on a specific mechanism of action rather than on a general toxic effect.Compounds pendency  and all L. crassum and L. laevigatum led to the identification of some novel cembranoid derivatives, including laevigatol B (1), which were evaluated for their anti-inflammatory and cytotoxic activities, as well as full assignment of all NMR data is reported [T. brucei, laevigatol B (1) exhibited inhibitory activity with an EC50 value of 5.34 ± 0.47 μM (1.69 ± 0.12 µg/mL). Although its activity was considerably weaker than that of the positive control (pentamidine), laevigatol B (1) did not display cytotoxicity against both human cell lines, and thus showed a certain degree of selectivity relative (50 = 0.36 ± 0.10 μg/mL) of this soft coral was more active than laevigatol B (1), suggesting the presence of other active derivatives, which could act individually or by synergy with laevigatol B (1). It is interesting to note in this respect that inhibition of parasites responsible for NTDs was already reported for cembrane-type diterpenes from soft coral species [et al. reported that the cembranoid serratol obtained from the plant Boswellia serrata was active against T. brucei rhodesiense and Plasmodium falciparum  [Our previous work on the constituents of the soft corals reported . In the relative . However species . Similarmalaria) . Further2‒5), (24S)-ergost-4-ene-3-one  and astropectenol A  showed stronger activity than that of cholest-8-ene-3β,5α,6β,7α-tetraol  and 5α-cholest-8(14)-ene-3β,7α-diol . This observation suggested that the presence of a ketone and/or aldehyde group might play an important role in their anti-trypanosomal activities. This is in agreement with recent reports of the antiparasitic inhibitory activities of other steroids  [1‒4 possess significant activity against T. brucei suggests that these compounds or related steroids might be interesting for further investigations for potential activity against related trypanosomatid human parasites.Comparing the observed effects between the structurally similar steroid derivatives  ,21,22. O1‒5 also for growth inhibitory activity against the intracellular form of T. cruzi, the causative agent of Chagas’ disease (American trypanosomiasis). Quite interestingly, in this assay, only the cembranoid diterpene 1 (laevigatol B) and steroid 5 (5α-cholest-8(14)-ene-3β,7α-diol) showed significant activity. As shown in 1 and 5 in terms of cell shapes and number at a concentration of 50.0 μM whereas both lead to a significant reduction of the number of intracellular parasites  compared to dimethyl sulfoxide (DMSO) control. Interestingly, the steroid 5α-cholest-8(14)-ene-3β,7α-diol (5) which was inactive against T. brucei, showed superior activity. Compound 5 decreased parasite survival by 97.1%, compared to the DMSO control while the cell number of the host cells was not significantly reduced. The diterpene laevigatol B (1) showed reduction of parasites growth to 84.8%. However, 1 also caused a reduction of the host cells by 33%, comparable to the DMSO control .We therefore tested compounds  control C. Since o 100 μM , it appeTo the best of our knowledge, no previous reports regarding the activity of the extracts and/or compounds derived from echinoderm species against parasites responsible for NTDs could be found .Lobophytum crassum, L.laevigatum, and Sinularia dissecta) and echinoderms (Astropecten polyacanthus and Diadema savignyi) were collected at Quangtri, Khanhhoa, Hue, and Haiphong, Vietnam, from April 2010 to May 2013, identified by Nguyen Thi My Ngan, M.Sc. (Institute of Oceanography) and Prof. Do Cong Thung . A list of these samples can be found in earlier publications [The samples of the soft corals  were purchased from Sigma ; fetal bovine serum (FBS), HMI-9 medium, streptomycin/penicillin, HEPES, RPMI medium were purchased from Gibco ; Draq5 was purchased from Biostatus ; 384 well assay plates were purchased from Griener .Resazurin sodium salts, adenine, biotin, NaHCOinvacuo at ambient temperature for 24 h. High purity compounds (>96%) were previously isolated and their structures elucidated from the methanolic extracts of the Vietnamese soft corals  and echinoderms (A. polycanthus and D. savignyi) [Each powdered material sample 150‒250 g) was extracted with MeOH (1 L) in glass jars (2 L) with screw-on lids. The methanolic extracts were filtered, combined, and concentrated using a rotary vacuum evaporator at a temperature about 40 °C and then further dried avignyi) ,15,16,170‒250 g wT. brucei strain 427 bloodstream forms were cultivated at 37 °C with 5% CO2 in HMI-9 medium supplemented with 10% FBS. Cultivated parasites  were seeded in 384 well plates with the natural compounds and incubated for 3 days. After the incubation, the parasites were exposed to 120 µM of resazurin sodium salt (10.0 µL of a 720 µM solution in water) for 5 h. Then, the parasites were fixed with 4% PFA and the assay plates were read by Victor 3™  at 530 nmEx/590 nmEm. Pentamidine was used as a reference drug and DMSO 0.5% was used as a drug-negative control.T. cruzi Y strain was performed as previously described [, T. cruzi parasites were maintained in form of Tissue Culture Trypomastigote (TCT) by circulation in LLC-MK2 cell line . Seven days after the infection, the supernatant containing TCT parasites was collected and used to reinfect new cultures of LLC-MK2. The U2OS human cell , cultivated in DMEM-high glucose media supplemented with 10% heat inactivated FBS, was used for the in vitro assay preparation. The cells were infected with the TCT form of T. cruzi. A homogeneous mixture of parasites and cells in DMEM-low glucose media, supplemented with 2% heat inactivated FBS, and was seeded into 384-well plates at 50.0 µL/well. The test compounds  were added in 10.0 µL of 6% DMSO  and the cells were incubated for 48 h at 37 °C, 5% CO2. After the incubation, the cells and parasites were fixed with 4% PFA and stained with 5.0 µM Draq5. Fluorescent images were acquired from each assay well using an Operetta microscope (Perkin Elmer).The assay for activity against escribed . Briefly2 in Dulbecco’s modified eagle medium containing 10% FBS. Both cell types were plated individually in 384 well plates for testing the natural compounds at 3-fold dilution from 100 µM in 10-points concentration and incubated for 3 days. To determine viability, 10.0 µL of a 280 µM solution of resazurin sodium salt in water was added to the cells in the assay plate for at least 5 h  and resazurin reduction was measured with a Victor 3TM fluorimeter (PerkinElmer) at 530 nmEx/590 nmEm.HEK293T and HepG2 cells were cultured at 37 °C with 5% COT. cruzi screening were subjected to in-house software to quantify cell numbers, parasite numbers, and infection ratio as previously described [T. brucei and T. cruzi assays, 83.0 µM of chlorpromazine for cytotoxicity, and DMSO as 0% drug activity for negative control). Dose response curves were fitted by sigmoidal dose-response with variable slope, equation described as Y = Bottom + (Top − Bottom)/(1 + 10(LogEC5 − X) − HillSlpe) using GraphPad Prism 6 Software .The acquired images from the high content intracellular escribed . Each co1‒5 of trypanosomal growth from the marine soft corals and echinoderms. While compounds 1‒4 showed activity against bloodstream forms of T. brucei, only the cembranoid diterpene 1 and steroid 5 were active against intracellular amastigote forms of T. cruzi. Since none of the compounds displayed any significant cytotoxicity, these and related compounds may open a new avenue for future drug development against African sleeping sickness and/or Chagas’ disease. It should be noted at this point, that the strain of T. brucei studied here belongs to subsp. brucei which is not human pathogenic but responsible for trypanosomiasis in cattle. Further investigations with human pathogenic T. brucei (subsp. gambiense and/or rhodesiense) are therefore warranted. Subsequent studies will also include the assessment of underlying mechanisms of action of these compounds and evaluation of their in vivo efficacy in animal models, which could not be performed in the current study due to low available amounts of the isolated compounds.In conclusions, we have identified five inhibitors


Ribosomal DNA has been a reliable source of taxonomic and phylogenetic markers due to its high copy number in the genome and stable variation with few polymorphisms due to the homogenizing effect of concerted evolution. Typically specific regions are amplified through polymerase chain reaction (PCR) with multiple primer pairs that generate often incomplete and overlapping regions between adjacent segments of 18S, ITS1, 5.8S, ITS2, and 28S rDNA nucleotide sequences when combined in tandem. To improve the efficiency of this effort, a strategy for generating all these molecular sequences at once through PCR amplification of a large ribosomal 3.3 to 4.2 kb DNA target was developed using primer 18S-CL-F3 paired with D3B or a new alternative 28S PCR primer (28S-CL-R) and other well-positioned and ribosomal-specific sequencing primers . The D1 region between ITS2 and 28S boundaries and the flanking sequence between 18S and ITS1 boundaries were fully revealed in this large nucleotide segment. To demonstrate the value of this strategy, the long rDNA segment was amplified and directly sequenced in 17 agriculturally important nematodes from the Tylenchida, Aphelenchida, and Dorylaimida. The primers and their positions may be employed with traditional Sanger sequencing and with next-generation sequencing reagents and protocols. Caenorhabditis spp., for instance, the rDNA copy number was estimated to be as many as 56 to 323 copies within their genomes  is arranged in tandem repeat arrays in the genome. Each repeat unit consists of one copy of small subunit (SSU) 18S, internal transcribed spacers (ITS1 and ITS2), 5.8S, and large subunit (LSU) 28S rDNA, and is separated by an external transcribed spacer (EST) and an intergenic spacer (IGS) . The cop genomes . Second, genomes . These t genomes . As a re genomes , one pai genomes , and one genomes . This muHeterodera spp. and Meloidogyne incognita and live adult nematodes from other taxa described in 2, 50 mM KCl, 0.45% TWEEN 20, and 0.05% gelatin, TM thermal cycler . This rapid physical disruption was repeated once. Then the tube was subjected to the third cycle of freezing in liquid nitrogen for 10 to 15 sec and then was slow-thawed at room temperature. The thawed sample was lysed with 1 µl of proteinase K  at 60°C for 60 min, followed by 95°C for 15 min to deactivate the proteinase K. At least three single nematodes from each taxon were picked for the individual DNA extraction. All resulting lysates were stored in a −20°C freezer until needed. The DNA extracts from Ditylenchus, Ecumenicus, and Radopholus were prepared previously by using mechanical lysis  buffer: 2.5 µl; dNTP mix, 2.0 mM each: 2.5 µl; 10 µM forward primer: 0.75 µl; 10 µM reverse primer: 0.75 µl; 0.625U DreamTaqTM Hot Start DNA Polymerase 5 U/µl: 0.125 µl, assembled per manufacturer’s manual] containing the primer pairs 18S-CL-F3 and either D3B or 28S-CL-R (a) at 57°C or 50°C  and then treated with ExoSAP-IT reagent  according to manufacturers’ protocols. Direct DNA sequencing was performed bidirectionally with multiple primers (Each PCR reaction was prepared with 2 µl of DNA extract and 23 µl of the PCR master mix [H28S-CL-R  and 2. P or 50°C  for 45 s primers  and an A primers  were newHelicotylenchus sp. 104G36 (MK292128), Heterodera orientalis 104F80 (MK292130), Hoplolaimus sp. 104G35 (MK292131), Meloidogyne incognita Me47 (MK292132), Pratylenchus scribneri Pr1(MK292133), and Xiphinema sp. 104F83 (MK292136) with high coverage in each base were deposited to GenBank. Additionally, another 11 agriculturally important nematodes were also tested by using the same approach described above when they became available, and the resulting sequences with Accession No. MK292123 for Aphelenchoides fragariae 104J12, MK292121 for Bursaphelenchus sp.104J7, MK292122 for Bursaphelenchus xylophilus104H46, MK292124 for Crossonema sp.104G38, MK292125 for Ditylenchus dipsaci 85A5, MK292126 for Ditylenchus sp. 85C1, MK292127 for Ecumenicus sp.85G11, MK292129 for Heterodera glycines H20, MK292138 for Litylenchus sp. 104H88, MK292134 for Radopholus similis 31G1, and MK292135 for Xiphinema sp. 06D2 were deposited to GenBank as well.The forward PCR primer for this long rDNA target was 18S-CL-F3, the reverse primer was D3B and a newly designed 28S primer, 28S-CL-R, was applied as well . Figure An approximate base-pair match between primer and template DNA is required for Taq DNA polymerase to begin an efficient PCR amplification cycle . The taxAlthough this long rDNA target was amplified successfully with a traditional Taq DNA polymerase used in the single primer pair approach, it should be noted that Taq DNA polymerase lacks 3′-5′ exonuclease activity and is incapable of proofreading any misincorporated nucleotides during PCR . This inIn this study, we demonstrated the PCR amplification of this 3.3 to 4.2 kb rDNA in 17 agriculturally important nematodes using the single primer pair approach. The taxonomic coverage by these two single primer pairs revealed in this study suggests that they may also be valid for other plant-parasitic nematode taxa in Tylenchida and Dorylaimida. Additionally, this ability was seen in several taxa in Rhabditida with the 18S-CL-F3 and 28S-CL-R pair (data not shown). This study also provides the internal ribosomal sequencing primers that are well positioned in the target rDNA  with hig


The photoluminescence spectrum presents a broad emission band peaking at 460 nm with a bandwidth of nearly 90 nm. By optimizing the Ce3+ concentration, an internal quantum efficiency (IQE) as high as 76% was achieved. Furthermore, 86% of the room-temperature emission intensity is still maintained at 150 °C, indicating a good thermal stability and practicality. A series of white LEDs were fabricated based on 405 nm chips coated with a blend of the new blue phosphor with the commercial yellow and red phosphors. High color rendering indexes (≥90) were achieved while the correlated color temperature was tuneable in the range of 3094 to 8990 K. These results suggest that SrLu2O4:Ce3+ can be utilized as a blue-emitting phosphor in NUV based white LEDs.Blue-emitting phosphors for near ultraviolet (NUV) based tri-color RGB phosphor blend converted white light emitting diodes (LEDs) have been extensively investigated in the past few years. LED chip peaked near 400 nm is the most efficient among the NUV chips currently. However, most of blue phosphors show inefficient excitation around 400 nm. Herein, a novel blue phosphor SrLu Up to now, the most commonly used method to get white light based on monochrome LED is to combine a yellow-emitting phosphor such as cerium substituted yttrium aluminum garnet (Y3Al5O12: Ce3+) with a blue (InGa)N LED chip2. However, the inconsistency of the aging characteristics of the blue chip and the phosphor can lead to the instability of the white light which is generated by the combination of the blue light from the chip and the yellow light from the phosphor. Furthermore, this type of white LED emits little red light and therefore has a low color rendering index8. To resolve this problem, one can use an alternative method to obtain stable white light by a combination of a red-green-blue (RGB) phosphor blend with a near ultraviolet (NUV) LED chip10. In this method, the visible components of the white light are generated only by phosphors, exhibiting low color point variation against the forward-bias currents. In the NUV chips, near 400 nm chip is more attractive because of the highest energy conversion efficiency. As a result, this approach requires all three RGB phosphors to have efficient excitation around 400 nm to maximize the device’s efficiency.White LEDs have been widely deployed commercially on solid-state lighting, because of their superior efficiency and long life4: Eu2+11, Ba3LaNa(PO4)3F: Eu2+12, BaHfSi3O9: Eu2+13, Ca2LuScZrAl2GeO12: Ce3+14, Ca3Hf2SiAl2O12: Ce3+15, Ca3Zr2SiGa2O12: Ce3+16, CaLaGa3S6O: Ce3+17, Ca2B5O9Br: Eu2+18, and Na3–2xSc2(PO4)3: xEu2+19. But few of them could match well with near 400 nm chip and have high thermal stability at the same time. Therefore, it is essential to develop novel blue-emitting phosphors showing both excellent thermal stability and high quantum efficiency.Some blue-emitting phosphors as candidates for NUV (~ 400 nm) chip excitation have been reported, such as LiCaPO2O4 , with the space group Pnam have been intensively studied for their magnetic properties22. Selected SrLn2O4 as hosts, Eu3+-doped SrLu2O4 and SrGd2O4 luminescent materials have been studied and considered as promising red phosphors in solid state lighting devices24. Eu2+ doped SrLu2O4 exhibited a broad red emission centered at 610 nm, but it could be applied in optical temperature sensor instead of white LED because of its strong thermal quenching25.The rare-earth strontium oxides, SrLn2O4: Ce3+ prepared by solid state reaction. Photoluminescence (PL) properties and temperature dependence of the new phosphor are studied. The new phosphor shows efficient blue emission with the luminescence excitation band matching well with near 400 nm NUV chip. Besides, the phosphor also has high thermal stability with 86% of its room temperature emission intensity remained at 150 °C. White LEDs with high color rendering index (CRI) at different correlated color temperature (CCT) were fabricated based on NUV chips coated with blends of the new blue phosphor with the commercial yellow and red phosphors.In this paper, we report to our knowledge for the first time, a novel blue phosphor of SrLux2-O4: xCe3+(x = 0.0005-0.008) were characterized by XRD at room temperature. These samples all exhibit a single phase similar with the SrLu2O4 (JCPDS# 32-1242) crystal structure in Fig. 3+. A slight shift of peak at 64.3° can be noticed. When x equals 0.01, peak shifts to bigger angle and it shifts to minor angle while x is more than 0.004. It can also be explained by Bragg equation:The phase purities and the crystal structures of the as-prepared powder samples SrLu2O4 structure, the ion radius of Ce3+ is between Sr2+ and Lu3+ which means it might take both sites. At low concentrations, Ce3+ tend to occupy Sr2+ sites. When x equals 0.01, Ce3+ occupied more Sr2+ than Lu3+ sites and represented shifting to bigger angle. While x is more than 0.004, Ce3+ occupied more Lu3+ than Sr2+ sites and represented shifting to minor angle. In this work, all the samples were synthesized based on the substitution of Ce3+ for Lu3+ by charge balance. However, it does not follow that the Ce3+ only occupies the Lu3+ site as mentioned above.Peak shifting to minor angle means sites are occupied by bigger ions. In SrLu2O4 is obtained according to the JCPDS cord using Diamond software, shown in Fig. In order to further understand the microstructure of the as-prepared samples, detailed Rietveld refinements and lattice parameters are performed in Fig. 2O4: 0.2% Ce3+ unwashed and washed, respectively. It can be seen that small particles were washed away by alcohol and the particle was about 3 µm. Figures 2O4: 0.2% Ce3+ phosphors under bright field light and 405-nm excitation, respectively. The size of particles in micrographs agrees with those in SEM images.Figure em = 460 nm) and PL (λex = 390 nm) spectra of SrLu2O4: 0.2% Ce3+ phosphors. The PLE spectrum of SrLu2O4: Ce3+ in the UV region from 250 to 450 nm contains four distinctive bands peaked at 265 nm, 306 nm, 359 nm and 405 nm mainly ascribed to the allowed 4f-5d transitions of Ce3+26. To obtain entire PL spectrum, we chose 390 nm as the excitation wavelength to avoid the effect of excitation light. The PL spectrum exhibits a strong blue emission peak at 460 nm with a full width at half maximum (FWHM) of approximately 90 nm. The asymmetric emission band can be well fitted with two Gaussian bands peaking at 20253 cm−1 and 22165 cm−1. Their energy difference is about 1912 cm−1, being consistent with the energy separation between the 2F7/2 and the 2F5/2 sub-states of the ground state of Ce3+ (~2000 cm−1)27.IQEs were measured using a spectral on-coated integrating sphere and the measured results show that the IQE is 76.1%. The optimization of Ce3+ concentration in SrLu2O4 was performed by studying the PL spectra of SrLux2−O4: xCe3+ for different Ce3+ concentrations , as shown in Fig. 3+. Further increasing Ce3+ concentration leads to PL decreasing due to the well-known concentration quenching effect. The concentration quenching originates from the larger probability of energy loss at a killer centre due to excitation energy migration among Ce3+ ions.Figure c), defined as an average distance between Ce3+ ions shorter than which luminescence quenching occurs, can be estimated from geometrical consideration by following formula28:c represents the critical concentration of Ce3+ ions, and N represents the number of sites that can be replaced by Ce3+ in the unit cell. In this study, V = 391.339 Å3, N = 12, and Xc = 0.002 for SrLu2O4: Ce3+. Thus, the Rc of Ce3+ ions were calculated to be 31.46 Å. The value is much larger than 5 Å, indicating that exchange interaction is not responsible and the electric multipolar−multipolar interaction should be suitable for the ET of this luminescence center29.Here, the critical quenching distance  versus log(x) can be fitted linearly with a slope −(θ/3) equal to −2.02. The value of θ is determined to be 6.06, which approximates to 6, implying that the concentration quenching of Ce3+ ions in the SrLu2O4: Ce3+ mainly results from the electric dipole-dipole interactions between Ce3+ ions. The large critical quenching distance might be explained as follows. Ce3+ ions may occupy Sr2+ and Lu3+ sites to form two luminescence centers (Ce1 and Ce2). We found that there was another emission band centered at 600 nm upon 480-nm excitation, as shown in Figures The energy transfer mechanism among Ce1.998O4: 0.002Ce3+ was also studied. This new blue phosphor, SrLu1.998O4: 0.002Ce3+, shows a stability in the luminescence color. As shown in Fig. 32I0 is the PL intensity at 0 K, here it is treated as the one at room temperature if the PL intensity is stable below room temperature, I(T) is the PL intensity at a given temperature T, A is a constant, Ea is the activation energy for thermal quenching, and kB is the Boltzmann constant. The experimental data are well fitted using Eq. , 2SiO4: Eu2+ (yellow) and Sr2Si5N8:Eu2+ (red) phosphors, the white LEDs based on NUV 405-nm chips have been fabricated and the digital images are shown in Fig. Combined with SrLu1.998O4:0.002Ce3+, commercial blue phosphor BAM and fabricated white LEDs  to carry on the conventional high temperature/high humidity (85 C/85RH) tests to prove its reliability. As shown in Fig. We chose SrLu2O4: Ce3+ phosphors have promising applications for white-light NUV LEDs.All these results indicate that SrLu3+ doped SrLu2O4 phosphor was synthesized via high-temperature solid-state reaction method. The XRD patterns indicated the purity of the crystal phase for the as-prepared samples. Under UV excitation at 405 nm, the PL spectrum of SrLu1.998O4: 0.002Ce3+ sample exhibits a broad blue emission band peaked at 460 nm with a high IQE of 76.1%. By changing the ratios of SrLu2O4: Ce3+, yellow-emitting 2SiO4: Eu2+, and red-emitting Sr2Si5N8: Eu2+ phosphors, a series of white NUV LEDs with excellent color rendering index (Ra ≥ 90) could be fabricated, and the correlated color temperatures were 3094 K, 4393 K, 4818 K and 8990 K, respectively. All results indicate that SrLu2O4: Ce3+ can be promisingly used as an ultraviolet-convertible blue-emitting phosphor.The Ce2O4: Ce3+ samples were synthesized by traditional high-temperature solid-state reaction. The constituent oxides and carbonates, Sr2CO3 (A.R.), Lu2O3 (99.99%), CeO2 (99.99%) were employed as the raw materials. The stoichiometric weighed powder according to the formula of SrLux2-O4: xCe3+ was mixed in an agate mortar and placed in an alumina crucible. This crucible was heated at 1600 °C for 6 hours in a reducing atmosphere (95%N2/5%H2), to reduce Ce4+ to Ce3+. After sintering, the powders were furnace-cooled naturally down to room temperature (RT). Finally, the as-prepared powders were washed with alcohol three times and dried at 60 °C for 6 hours in a drying oven to obtain final phosphors. The NUV 405-nm chips we used were produced by Guangsheng Semiconductor Technology Co., Ltd. (chip size is 1.143 mm × 1.143 mm and efficiency is 40%)SrLu3+ were measured by FL900 fluorometer with a Xenon lamp . The fluorescence micrographs and temperature-dependent PL spectra were carried out on a fluorescence microscopy . Internal quantum efficiency (IQE), i.e. the number ratio of the photon emitted to the photon absorbed, were measured directly by the absolute PL quantum yield measurement system . The high temperature/high humidity (85 C/85RH) condition was provided by Programmable Temperature & Humidity Chamber . The photoelectric properties of the fabricated white LEDs were measured by HAAS 2000 photoelectric measuring system . The forward bias current was 20 mA. All the measurements were conducted at room temperature unless mentioned specially.X-ray diffraction (XRD) patterns were performed by a powder diffractometer . The XRD data were collected in range of 15 to 75 degree (2θ) with count time of 2 s/step. The PL and photoluminescence excitation (PLE) spectra of CeSupplementary Information


Nanocomposites of g-C3N4 supported by CdSe QDs (CdSe QDs/g-C3N4 nanosheets) were fabricated through an electrostatic interaction route in an aqueous solution. The nanocomposites were characterized by X-ray photoelectron spectroscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy. Results showed that the g-C3N4 nanosheets were decorated randomly by CdSe QDs, with average diameter of approximately 7 nm. The feasibility of the FRET system as a sensor was demonstrated by Hg (II) detection in water. At pH 7, a linear relationship was observed between the fluorescence intensity and the concentration of Hg (II) (0–32 nmol/L), with a detection limit of 5.3 nmol/L. The new detection method was proven to be sensitive for detecting Hg2+ in water solutions. Moreover, the method showed high selectivity for Hg2+ over several metal ions, including Na+, Mg2+, Ca2+, Pb2+, Cr3+, Cd2+, Zn2+, and Cu2+. The CdSe QDs/g-C3N4 nanosheet conjugate exhibited desirable long-term stability and reversibility as a novel FRET sensor. The novel FRET-based fluorescence detection provided an attractive assay platform for quantifying Hg2+ in complex water solutions.In the presence of Hg Many fluorescent sensors for Hg2+ had been reported [2+ detection. Moreover, different FRET systems had been developed for the detection for Hg2+ [3N4 nanosheets had attracted widespread interest [3N4 nanosheets have been applied as sensors, a FRET detection system with g-C3N4 nanosheets and CdSe QDs for metal ions has not been reported. FRET-based fluorescence sensing systems offer multiple advantages [The main cause of mercury poisoning in humans was polluted natural waters . Hg2+ ioisorders . Semiconreported –8. For ereported  developefor Hg2+ –12. Notafor Hg2+ –15. Amoninterest , 17. Altvantages .3N4 nanosheets and CdSe QDs particles as vehicles. The proposed mechanism was illustrated in Fig. In the present study, a new FRET-based fluorescence sensor was developed to detect mercury ions in aqueous media by using g-C2) was purchased from Tong Ren Chemical Research Institute . Urea and CdSe QDs were purchased from Aladdin Reagent Company . Other reagents and chemicals were of analytical reagent grade and used without further purification. All solutions were prepared using purified water from a Milli-Q gradient water purification system .Mercury (II) chloride (HgClAn X-ray diffractometer (Rigaku D/max-2400) was used to obtain diffraction patterns. Ultraviolet–visible (UV–vis) spectra were recorded on a UV–vis 800 spectrophotometer at room temperature. Fourier-transform infrared (FTIR) spectra were recorded on a Nicolet-nexus670 spectrometer using KBr. Fluorescence measurements were performed at room temperature with an RF-5301PC fluorescence spectrometer. X-ray photoelectron spectroscopy (XPS) measurements were performed using a multifunctional spectrometer (Thermo Scientific).3N4 . The CdSe QDs/g-C3N4 nanosheet conjugate emission spectra were recorded. All samples were excited at 334 nm, which was near the minimal acceptor absorption.In a typical procedure, g-Cs report ) was dis2+ was quenched at room temperature in water. During a typical operation, 10 μL of the CdSe QDs/g-C3N4 nanosheet conjugates was added to 3 mL of ultrapure water, and then the calculated amount of Hg2+ was added. The emission spectra of the CdSe QDs/g-C3N4 nanosheet conjugates were recorded 2 mins later at room temperature.Hg+, Mg2+, Ca2+, Pb2+, Cr3+, Cd2+, Zn2+, and Cu2+) was studied through fluorescence spectroscopy. Studies were carried out using the CdSe QDs/g-C3N4 nanosheet conjugates emitting at 450 nm. The conjugate solution was placed in a 1-cm optical path quartz fluorescence cuvette. Fluorescence intensity was measured at emission wavelength of 450 nm under excitation wavelength of 334 nm in the presence of each possible interference (32 nM). Competition assays were also performed for all the possible interferences previously analyzed. For competition experiments, 32 nM Hg2+ aqueous solutions were prepared.The response of the FRET nanoprobe to other metal ions  peak of g-C3N4. The small peak at 13.1° corresponded to the periodic in-plain structural packing feature within the sheets [3N4 nanosheets. The XPS spectrum in Fig. −1 was attributed to the vibration of the triazine ring. The peaks around 1000 cm−1 represented the stretching modes of CN heterocycles, and the peak at 1800 cm−1 corresponded to C–NH–C. The peaks at 300–3600 cm−1 corresponded to N–H and O–H stretching vibrations [The structure and morphology of g-Cw layers . Figure e sheets , 21. XPSbrations .Fig. 2Ch3N4 nanosheets. As shown in Fig. 3N4 nanosheets at 14–16 nm ; the results were shown in Fig. 2+ increased obviously, while the fluorescence intensity of other metal ions changed slightly or remains the same. These results indicated that the FRET sensor showed more selectivity than the others , as shown in the inset of Fig. 3N4 nanosheets and CdSe QDs displayed no obvious quenching response to other metal ions apart from Hg2+, which suggested a relatively high selectivity for this method.To study the practicability of the FRET sensor, the CdSe QDs/g-C3N4 nanosheet-based sensing system toward Hg2+ ion in the presence of alkali, alkaline earth, and other transition metal ions was shown in Table 2+, which indicated that the interference of these coexisting ions on the Hg2+ sensor was negligible.The other coexisting cations that affect the detection of mercury ion were detected as well. The response of the CdSe QDs/g-C3N4 nanosheets remained above 92% of the initial efficiency though they were stored at ambient environment. The results indicated that the CdSe QDs/g-C3N4 nanosheets as FRET sensors had good long-term stability.In addition, long-term stability is a superior property of sensors. The absorbance and the fluorescence during the continuous investigation every 3 days within 2 weeks indicated that the activity of CdSe QDs/g-C2+ (results are listed in Table 3N4 nanosheet fluorescence probe based on FRET with the concentration of Hg (II) in the range of 0–32 nmol/L at pH = 7 exhibited a limit of detection at 5.3 nmol/L. Thus, our method obtained a superior detection limit and linear range.Compared with previous reports concerning fluorescence assays for Hg3N4 nanosheets as a FRET sensor successfully provided a good platform for detecting Hg2+ in real samples because of their sensitivity and selectivity. Well, lake, and tap waters were selected as real samples for analysis in which the recovery of Hg2+ were in the range of 95.4–101.6%  in the presence of Hg2+ for the first time.Fluorescence resonance energy transfer (FRET) system was constructed between CdTe quantum dots (QDs) (donor) and g-C3N4 supported by CdSe QDs (CdSe QDs /g-C3N4) were fabricated through a simple electrostatic interaction route in an aqueous solution.The nanocomposites of g-CThe feasibility of the FRET system as a sensor was demonstrated for detecting Hg (II) in water solution. At pH 7, a linear relationship was observed between the quenched fluorescence intensity of the concentration of Hg (II) in the range of 0–32 nmol/L. The detection limit was 5.3 nmol/L.2+ in complex water solutions.The novel FRET-based fluorescence detection may provide an attractive assay platform for quantifying Hg