Many studies have found that ruthenium complexes possess unique biochemical characteristics and inhibit tumor growth or metastasis.2b, which has both antitumor and antimetastatic properties and targets tumor sites through the enhanced permeability and retention (EPR) effect and transferrin/transferrin receptor (TF/TFR) interaction. The candidate 2b is composed of ruthenium-complexed carboline acid and four chloride ions. In vitro, 2b triggered DNA cleavage and thus blocked cell cycle progression and induced apoptosis via the PARP/ATM pathway. In vivo,2b inhibited not only Lewis lung cancer (LLC) tumor growth but also lung metastasis. We detected apoptosis and decreased CD31 expression in tumor tissues, and ruthenium accumulated in the primary tumor tissue of C57BL/6 mice implanted with LLC cells.Here, we report the novel dual-targeting ruthenium candidate 2b targets tumors, inhibits tumor growth and prevents lung metastasis.Thus, we conclude that  Metal-based antitumor drugs, such as cisplatin, have been extensively studied over the past few decades . ResearcCarboline alkaloid is an active ingredient extracted from traditional Chinese medicine. Our group has conducted in-depth research on its antitumor and other activities , 17. ManAlthough platinum (IV) prodrugs have great potential to kill tumor cells and reduce side effects , 24, few2 and complexes 2a and 2b on various cancer cell types and normal cells using MTT assays. We determined the self-assembly and interaction of 2b with DNA and TF to explore dual-targeting functions. We also determined the localization of ruthenium via inductively coupled plasma mass spectrometry (ICP-MS), protein expression in A549 cancer cells via western blotting, and effects of 2b on apoptosis and the cell cycle to further explain its mechanism. In vivo, we evaluated the effects of 2b on primary tumor growth and lung metastasis in C57BL/6 mice implanted with Lewis lung carcinoma (LLC) cells. We also assessed apoptosis in tumor tissues via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and platelet endothelial cell adhesion molecule-1 (CD31) expression via immunohistochemistry.Our aim was to obtain a dual-targeted ruthenium complex with both antitumor and antimetastatic properties. This complex targets tumor sites through both the enhanced permeability and retention (EPR) effect and TF/TFR interaction. We designed and synthesized the ruthenium complex 2b (ruthenium (IV)) Fig. a. To tes1H NMR and 13C NMR spectroscopy  in the FTIR spectrum of ligand 2 shifted to 1621 cm−1 and changed to a broad peak in the FTIR spectrum of complex 2b. The final complexes were characterized using mass spectra.We deduced the compound structures Fig. a from th2a and 2b were air stable and water soluble, while the carboline derivative 2 was only slightly water soluble  and scanning electron microscopy (SEM)  Fig. b, c, theEM) Fig. d, and thEM) Fig. e. TEM anin water , 32. The2b essentially remained stable for 96 h, while the zeta potential of 2 gradually fell to 0 within 96 h. This indicates that the 2b nanoparticles were stable and dispersed in aqueous solution over 96 h. The zeta potential of 2 and 2b are approximately − 10 mV and − 15 mV, respectively, at pH 7.0. The negative zeta potential is likely due to the negative charge of the COO− groups [2b changed slightly but that of 2 decreased substantially. At a low pH of 2.0, 2 even showed a positive zeta potential but showed negative zeta potentials at higher pH values. This is consistent with previous nanoparticle studies [The zeta potential of − groups . As the  studies , 35.2b by measuring fluorescence quenching of hTF [2b increased , biomolecular quenching rate constant , binding constant  and the number of binding sites (n)  to complex g of hTF , 27, 36.sed Fig. g. We cal2b clearly confirmed binding of hTF to 2b  spectroscopy quenching of mplex 2b . With th2, 2a, 2b and NAMI-A on four lung cancer cell lines , a mouse sarcoma cell line (S180) and a normal liver cell line  polymerase (PARP), cleaved caspase 3, ataxia-telangiectasia mutated (ATM), gamma H2A histone family member X (γ-H2A.X), CDK1 and programmed cell death 1 ligand 1 (PDL1), in A549 lung cancer cells via western blotting , 21 . With an increasing concentration of 2b, the number of cells in G0/G1 phase decreased gradually, whereas the number of cells in G2/M phase increased gradually; the number of cells in S phase did not change significantly. Hence, we concluded that 2b is able to stall cells in G2/M phase.We studied the effect of ytometry , 22. Cel2b, the number of cells in Q2 and Q4 gradually increased. We also observed apoptotic cells using laser confocal microscopy  according to the fluorescence intensity of Annexin V-fluorescein isothiocyanate  and 2b (5.0 mg/kg) in inhibiting primary tumor growth and lung metastasis.We studied the effect of LC cells , 42. Fig2b treatment groups and the vehicle group were not significantly different. We also determined the distribution of 2b in the organs of C57BL/6 mice [2b can target tumors in vivo.Platinum agents can decrease organ weights . The weiL/6 mice . Figure 2b in tumor tissues [2b treatment group was significantly increased compared with that in the vehicle and NAMI-A groups. This suggests that drug-induced apoptosis may be one of the reasons that 2b inhibits the growth of LLC tumors.Using TUNEL staining, we detected apoptosis induced by  tissues . As show2b significantly reduced CD31 expression. We conclude that 2b may exert its effects through the same mechanism as NAMI-A because they both inhibit tumor neovascularization to suppress tumor metastasis.As CD31 is a marker of vascular endothelial cells, we evaluated the neovascularization of tumors according to CD31 expression –15. As d2b [NAMI-A and 50 μM, 20 μM, or 10 μM 2b for 6 h. As shown in Fig. NAMI-A and 2b decreased tube formation at 6 h. In addition, 2b inhibited capillary tube formation in a dose-dependent manner.Capillary tube formation assays were used to assess the angiogenic activity of 2b , 45–47. 2b. At 30 min after administration, the Ru was distributed evenly in plasma and blood cells and reached a maximum plasma concentration at 2 h after administration, while the content in blood cells decreased to almost zero , aspartate aminotransferase (AST), blood urea nitrogen (Urea) or creatinine (Crea) levels.In the toxicity study, the heart, liver, spleen and kidney weights were significantly reduced in the cisplatin group, while the weights of organs in the 2b was designed to compensate for the shortcomings of low solubility and poor targeting of carbolines. In vitro, we compared the cytotoxic effects of ligand 2 and complex 2a and 2b on different cancer cell types and normal cells using MTT assays. Only 2b was able to selectively kill cancer cells. This partially validates the hypothesis that chloridion plays an important role in tumor-targeting therapy. The introduction of chloridion into carbolines yields amphiphilic structures to increase selectivity for cells. We determined the self-assembly properties and protein binding characteristics of 2b to evaluate its targeting characteristics. 2b remained stable for 96 h [2b is 50–100 nm, which is more favorable for targeting tumors than ligand 2 based on the EPR effect [2b strongly bound to TF [2b can target tumor tissues with TF as the vector [The nanoscale self-assembly properties of small molecule drugs are important in tumor-targeting therapy , 50, andfor 96 h ; and thend to TF . Becausee vector , 53.2b in A549 cells  The EPR effect and TF/TFR interaction with nanoparticles improve tumor targetability in vivo. The Ru content in tumors accounted for 33.4% of the total content  groups. As described in reference [2b was randomly distributed with a density of 0.5 g cm−3. A 15 000 ps simulation was performed on this system at 298 K using the NVT ensemble.To predict its nanostructure, we defined eference , in a cu2b and its ligand 2 in solution and in solid state, a DLS particle size analyzer, TEM and SEM were applied. The nanoparticles were prepared via the self-assembly method [To explore the nanoscale self-assembly properties of y method , 49, 56.2 and 2b  were dripped onto a formvar-coated copper grid. After thorough drying in air, the copper grid was kept in the dryer for 48 h. Then, the shape and size of the nanoparticles were observed with TEM and SEM. The details of this experiment are described in Additional file Aqueous The particle size and surface zeta potential of the particles were measured using a particle size analyzer. The compounds were dissolved in ultrapure water with 1% DMSO. The concentration of the compounds in solution was 0.01 mg/mL, and the pH of the solution was pH 2.0, pH 5.5 or pH 7.4. The details of this experiment are described in Additional file 6 viable tumor cells under the skin into the right armpit of the mouse. When the tumor size reached approximately 5 mm in diameter (days 7–10 after implantation), the mice were randomly divided into the following treatment groups: 2b , NAMI-A , and vehicle: 20% 2-hydroxypropyl-β-cyclodextrin . The drugs were administered by intraperitoneal injection, and mice were weighed daily. 24 h after the last injection, the mice were weighed and killed with ether anesthesia, and the organs and tumor were immediately obtained.Male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The study was approved by the Institutional Animal Care and Use Committee of Capital Medical University, and the ethics number is AEEI-2018-174. The animals were cared for humanely throughout the animal studies. Male C57BL/6 mice were 8 weeks old at the beginning of the in vivo tumor metastasis assay. LLC cells were subcutaneously injected to form solid tumors. The subcutaneous tumors were implanted by injecting 0.2 mL of normal saline (NS) containing 1 × 10Nanoparticle degradation assays were carried out with C57BL/6 mice implanted with LLC cell-derived tumors. Three mice in each group were sacrificed after 0.5 h, 1 h, 2 h, 8 h and 24 h. 2b was injected into the tail vein at a single dose of 5 mg/kg. Blood and organs were harvested immediately after the animals were sacrificed at the given time points. Blood was taken from the eye, added to tubes containing EDTA and centrifuged (2500 rpm for 5 min) to separate plasma and blood cells .In the toxicity study, 6 ICR mice implanted with S180 cell-derived tumors in each group were sacrificed on seven consecutive days. The drugs were administered via intraperitoneal injection at 1.0, 2.5 or 5.0 mg/kg/day, and the dose of cisplatin was 5 mg/kg. Blood was taken from the eye, added to tubes without anticoagulant and centrifuged (3000 rpm for 10 min at 4 °C) to obtain the serum. Serum Crea, Urea, ALT and AST levels were measured with a chemistry analyzer and the appropriate reagents . The heart, liver, spleen, kidneys, and brain were weighed.Additional file 1. Supplementary Information of a dual-targeting ruthenium nanodrug that inhibits primary tumor growth and lungmetastasis via the PARP/ATM pathway.


Cardiomyocyte‐specific deletion of S1pr1 during mouse development leads to ventricular noncompaction, with 44% of mutant mice surviving to adulthood. Adult survivors of embryonic cardiomyocyte S1pr1 deletion showed cardiac hypertrabeculation consistent with ventricular noncompaction. Surprisingly, systolic function in mutant mice was preserved through at least 1 year of age. Cardiac conduction was abnormal in cardiomyocyte S1pr1 mutant mice, with prolonged QRS intervals in mutants as compared with littermate control mice. Immunostaining of hearts from S1pr1 mutant embryos displayed a zone of intermediate Connexin 40 (Cx40) expression in the trabecular myocardium. However, we observed no significant differences in Cx40 and Connexin 43 immunostaining in hearts from adult survivors of embryonic cardiomyocyte S1pr1 deletion, which suggests normalized development of the ventricular conduction system in mutant mice. By contrast, the adult survivors of embryonic cardiomyocyte S1pr1 deletion showed increased cardiac fibrosis as compared with littermate controls. These results demonstrate that ventricular hypertrabeculation caused by embryonic deletion of cardiomyocyte S1pr1 correlates with cardiac fibrosis, which contributes to abnormal ventricular conduction. These results also reveal conduction abnormalities in the setting of hypertrabeculation with normal systolic function, which may be of clinical relevance in humans with ventricular hypertrabeculation.Sphingosine 1‐Phosphate receptor 1  displayed an abnormal expression pattern in Mlc2aCre/+; S1pr1f/− mutant embryos. However, Cx40 and contactin‐2 expression in adult mice did not differ from littermate controls, which suggests normalized development and maintenance of the ventricular conduction system. Finally, cardiomyocyte S1pr1 mutant mice demonstrated increased cardiac fibrosis that correlated with QRS prolongation. In adult mice that survived embryonic deletion of  This breeding strategy created Mlc2aCre/+; S1pr1f/− mutant mice that lack S1pr1 in cardiomyocytes, as well as S1pr1f/+, S1pr1f/−, and Mlc2aCre/+; S1pr1f/+ littermate controls , conditional sphingosine kinase 1 mice , and sphingosine kinase 2 global knockout mice . For Sphk1; Sphk2 cardiomyocyte deletion studies, we maintained Mlc2aCre/+ on the Sphk1+/−; Sphk2−/− background. Mlc2aCre/+; Sphk1+/−; Sphk2−/− males were bred with Sphk1f/f; Sphk2−/− females to generate Mlc2aCre/+; Sphk1f/−; Sphk2−/− with deletion of sphingosine kinases in cardiomyocytes  provided s Figure . Dr. Shas Figure .2.3All mouse procedures were approved by the Institutional Animal Care and Use Committee at Northwestern University. Both male and female mice were used between 6 and 12 months of age in adult mouse echocardiographic and electrocardiographic studies.2.4The morning of vaginal plug was defined as 0.5 days post coitus (dpc). Pregnant females were euthanized at 15.5 dpc. Embryos were removed to ice‐cold PBS, dissected away from the placenta, and snap frozen in OCT using liquid nitrogen‐cooled isopentane. For a subset of embryos, the heart was dissected out and frozen in OCT. In all cases, a portion of the embryo was removed for genotyping.2.5Adult mouse echocardiograms were recorded using a Vevo2100 echocardiography platform with a 550 MHz solid‐state probe (FujiFilm). Mice were anesthetized using 2% isoflurane, and anesthesia was maintained using 1% isoflurane during collection of echocardiograms. Mouse heart rate was maintained between 400 and 600 (bpm). Recordings were taken from both parasternal long‐axis and short‐axis views. Left ventricular wall thickness and internal dimensions during both systole and diastole were measured approximately midway through the chamber. Fractional shortening was calculated using measurement from three beats per recording. Compact and trabecular wall thickness were measured in diastole using Fiji  were recorded using the ECGenie platform . Mice were acclimated to the apparatus, then placed on the electrode pad for approximately 20 minutes. Mice were not anesthetized during the recording of ECGs. A consistent and representative subsection of 10–20 cardiac cycles of the ECG recordings was analyzed using the EzCG Signal Analysis Software.2.7Mice were anesthetized using 4% isoflurane. Once anesthetized, thoracotomy was performed, the heart was fully exposed, the inferior vena cava was severed to release blood, and 3–5 ml of cardioplegia  solution was perfused into the left ventricle (LV). The heart was removed, placed into an aluminum ladle, and snap frozen using liquid nitrogen. Frozen hearts were stored at −80°C until cryosectioning. For samples that underwent paraformaldehyde fixation, hearts were perfused with 4% paraformaldehyde in cardioplegia, dissected out of the thorax, fixed overnight in 4% paraformaldehyde in phosphate‐buffered saline, and then processed for paraffin embedding.2.8Microtome sections of 6 µm thickness and cryosections of 10 µm thickness were collected as frontal sections of the hearts. Sections selected for staining contained both ventricles and the mitral valve in order to include the VCS. For hematoxylin and eosin (H&E) staining, sections were rehydrated and stained using standard procedures. For wheat germ agglutinin (WGA) staining, sections were fixed using 4% paraformaldehyde and incubated with WGA Alexa Fluor 488 conjugate (ThermoFisher #W11261) at 5 µg/ml for 10 minutes. Immunostaining was performed as previously described  using silica beads and a homogenizer (BioSpec). Lysates were measured using the Pierce BCA assay kit (Thermo Scientific #23225) and normalized to 10 µg total protein. Lysates were separated using polyacrylamide gel electrophoresis and gel was transferred to nitrocellulose (NC) membranes. Membranes were blocked in 5% BSA prepared in 1x TBS‐T. After blocking, NC membranes were incubated with primary antibodies Cx40 , Connexin 43 , or Contactin 2 . Membranes were incubated with secondary antibody goat anti‐rabbit IgG‐HRP  or donkey anti‐goat IgG‐HRP . Signals were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific #34577). Total protein was determined using Memcode Reversible Protein Stain Kit (Thermo Scientific #24580).2.11p < 0.05 was considered statistically significant.All measurements were done blind to genotype. Differences were assessed using one‐way ANOVA, and post‐hoc individual comparisons were performed using the Bonferroni test. Correlation between QRS length and percent fibrosis was assessed using Pearson correlation coefficients. Genotype distributions were assessed using the chi‐square goodness of fit test. All statistical analyses were performed using GraphPad software. 33.1S1pr1 during embryonic development using a Cre recombinase driven by the promoter for Myl7 . To avoid germline excision in the setting of exogenous Cre expression during development, we maintained Mlc2aCre/+ on the S1pr1+/− background. Conditional S1pr1 mutant mice were generated in crosses of S1pr1f/f females with Mlc2aCre/+; S1pr1+/− males. This breeding strategy created Mlc2aCre/+; S1pr1f/− mutant mice that lack S1pr1 in cardiomyocytes, as well as S1pr1f/+, S1pr1f/−, and Mlc2aCre/+; S1pr1f/+ littermate controls  of mutant mice died in the perinatal period, but the surviving cardiomyocyte S1pr1 mutant animals had a normal lifespan of 18 months or longer  survived to weaning , and sphingosine kinase‐2 (Sphk2); their protein products catalyze phosphorylation of sphingosine to S1P  interval, which reflects ventricular repolarization Figure . We also3.3Gja5). Furthermore, altered expression of Cx40 and Cx43 has been observed in mouse and human cardiomyopathies with aberrant ventricular conduction  immunostaining and WGA‐Alexa Fluor 488 staining. We detected low levels of Col1a1 and WGA in heart sections from S1pr1f/+ and S1pr1f/− mice, while sections from CreMlc2a/+; fS1pr1/+ mutant showed moderate levels of Col1a1 immunostaining  and endothelial‐specific deletion of Ino80 led to the same Cx40 expression pattern that we observed , a data set of 125,748 exome sequences, and 15,708 whole‐genome sequences from unrelated individuals, identified two unique frameshift variant alleles at amino acid positions 255 and 256  . Furthermore, missense variants identified in the NHLBI Exome Sequencing Project have been functionally assessed: the Arg120Pro variant  failed to activate S1P1 signaling or internalization in response to S1P, and Arg120 was found to be critical for S1P binding  was less strong than the correlation between fibrosis and QRS length  and S1pr1 dosage in cardiomyocytes. Furthermore, few adult mouse models for hypertrabeculation/noncompaction exist, and CreMlc2a/+; fS1pr1/− mutant mice represent a useful model to investigate this cardiomyopathy. The presence of conduction abnormalities in hypertrabeculated hearts, despite having normal systolic function, may be of clinical relevance for patients with hypertrabeculation.In conclusion, we report that mice that survive embryonic deletion of This work was supported in part by a grant from the Gilead Sciences Research Scholars Program in Cardiovascular Disease (L.D.W.). The funding source had no role in the design, collection and analysis of data, writing, or submission of the manuscript.R.J., M.K., and L.D.W. conceived and designed the research; R.J., M.K., J.W., D.F.L., B.L., and L.D.W. performed the experiments; R.J., M.K., D.F.L., and L.D.W. analyzed the data; R.J. and L.D.W. interpreted the results of experiments; R.J. and L.D.W. drafted the manuscript; R.J., M.K., J.W., D.F.L., B.L., J.C., and L.D.W. edited and revised the manuscript; R.J., M.K., J.W., D.F.L., B.L., J.C., and L.D.W. approved the final version of the manuscript.Tables S1–S3, Figures S1–S5Click here for additional data file.


Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring. Theblockers . Recent blockers ,7. Its pthe cell . The lonmembrane . AML hasn plasma .0) and the lowest excited singlet state (S1). This is achieved by the rotation of 2-chlorophenyl ring followed by the DHP fragment deformation. Suppression of such large amplitude motion in AML, for example by a viscous medium, leads to the fluorescence enhancement. When bound to a protein, the motion of the ligand is severely limited [Our calculations show that the fluorescence loss in aqueous solutions is caused by the nonradiative relaxation and proceeds through the conical intersection between the ground state  increases (Φ) and the lifetime (τ) of AML fluorescence were significantly higher in the presence of BSA or HSA than for a free drug ree drug . The sell of AML . To studglycerol . The batglycerol .η); their dielectric constants 32.6 and 42.5 respectively  and non-radiative (nrk) decay constants illustrate this effect with 0k remaining almost constant and decreasing nrk with increasing viscosity of the medium  the calculated molecular structure of AML is similar to the one obtained by single crystal X-ray analysis [1, from which it emits a photon and the fluorescence occurs. The molecular geometry of AML in S1 does not change significantly from the one in the S0 -AML in the 0–750 µM protein (BSA or HSA) solutions and observed the fluorescence intensity changes at the emission maximum (dK) were in a good agreement with previous studies where the tryptophan (Trp) fluorescence quenching was used for the binding constant determinations [λem (Trp) = 350 nm helps to minimize autofluorescence.The enhancement of AML fluorescence upon binding to BSA or HSA allowed us to determine the AML-protein binding affinities by fluorescence titration experiments. For that, we recorded the fluorescence spectra of 5 µM AML or -(−)-Bay K8644.12. (S)-(−)-Bay K8644 had no effect on cell staining by AML , ER-Tracker® Red (endoplasmic reticulum); and genetic markers: Golgi-emGFP, Rab7a-emGFP (late endosomes), Rab5a-emGFP (early endosomes) and LAMP1-emGFP (lysosomes). We treated living A7r5 cells with the respective marker and AML, and then visualized using a confocal Leica SP8 microscope. After 1 h incubation, the late endosomal and lysosomal labelling showed the co-occurrence of the cyan (AML) and green channel (emGFP) emissions with calculated Pearson’s co-localization coefficients (R) above threshold: R ~0.4 -(–)-amlodipine ((S)-AML) besylate (98 +%) was purchased from Carbosynth . (S)-(−)-Bay K8644 was purchased from Tocris Bioscience . BSA (98 +%), HSA (Fraction V) and Phosphate Buffered Saline tablets for 0.01 M phosphate buffer (PBS) were purchased from Merck, Darmstadt, Germany. All reagents were used without further purification. To prepare 20 mL of 10% BSA/HSA solutions in phosphate buffered saline , 2 g of protein was dissolved in a 20 mL volumetric flask with the previously prepared PBS. The protein solution was kept in a refrigerator until use.Racemic amlodipine besylate (97+%) was purchased from Alfa Aesar .  at room temperature (25 °C). Fluorescence emission of AML was recorded from 390 nm to 600 nm with excitation at 360 nm. The emission bandwidth was set to 10 nm, excitation bandwidth—7.5 or 10 nm, gain—100, number of flashes—20. The absorption spectra were recorded: from 300 to 430 nm. The background absorption of a glass bottom plate was measured in wells containing only buffer and subtracted from the spectra of the samples. The spectra were averaged from three individual experiments. The absorption spectra of AML in glycerol/methanol mixtures were recorded on a Varian Cary 4000 UV-Vis spectrophotometer in quartz cuvettes (3 mL) with a 1 cm path length. Fluorescence spectra of 5 µM AML in PBS solutions of different pH (3–9) were recorded on a Varian Cary Eclipse fluorescence spectrometer.Φ) were obtained on a Quantaurus-QY absolute PL quantum yield spectrometer  at ambient temperature (25 °C), excitation wavelength 370 nm; all measurements were performed in triplicates. Fluorescence lifetimes (τ) were measured with a Quantaurus-Tau fluorescence lifetime spectrometer . All measurements were performed in air-saturated solvents at ambient temperature. Radiative fluorescence lifetime (radτ) was calculated by the formula: radτ = τ/Φ.Absolute fluorescence quantum yields . All transient spectra were recorded using 10 × 10 mm quartz cells with a capacity of 4 mL and were bubbled for 30 min with argon before acquisition. All the experiments were conducted at room temperature.The molecular geometry of the compound and its spectra were calculated using the FireFly program package  partiall−1 and f is its oscillator strength. The rate of achieving the conical intersection via the barrier was calculated by the Arrhenius formula: −1 and A is the barrier height.The radiative rate constant was calculated by the formula: v/v% DMSO). The obtained solutions were incubated for 1 h at room temperature to ensure ligand binding to the proteins. Emission spectra of BSA and HSA solutions, as well as the spectra of BSA/HSA solutions containing AML or (S)-AML (5 µM), were measured on a multiwell plate reader as given in “General materials and methods” section; the emission wavelength step size was 1–2 nm. The fluorescence intensities at 450 nm were measured for the calculation of the AML fluorescence increase upon binding to BSA/HSA. The fluorescence increase of compounds in BSA or HSA in relative fluorescence units (RFU) was plotted vs. proteins concentrations. Binding constants were calculated using the software package GraphPad Prism version 8.3.1 . Y = (Bmax × X/(dK + X)) + off was used, where Bmax is the maximum specific binding.The tested compounds were dissolved in the PBS containing different concentrations of BSA or HSA  were cultured in DMEM/F-12  supplemented with GlutaMAX-1 (Thermo Fisher Scientific), 10% fetal bovine serum (FBS)  and 0.9% Penicillin/Streptomycin  in a humidified 5% CO2 incubator at 37 °C. In addition, we used a reduced antibiotic pressure for the cultivation of the cells; 100 µg/mL hygromycin B , 15 µg/mL Blasticidin , 0.4 µg/mL puromycin . To induce the expression of CaV1.2, 0.1 µg/mL doxycycline  was added 24 h before experiments.HEK-293 Ca2 atmosphere at 37 °C. Cells were split every 3–5 days or at confluence.A7r5 cells  were cultured in DMEM Medium supplemented with 10% FBS, 0.9% penicillin/streptomycin in a humidified 5% COHL-1 cells  were cultivated in Claycomb’s Medium (Sigma), supplemented with Glutamax, 10% fetal bovine serum, 0.9% penicillin/streptomycin and 0.1 mM noradrenaline (Sigma) on Fibronectin/Gelatine (Sigma) coated culture bottles or plates.2+ assay, HEK-293 CaV1.2 cells  or HL-1 cells  were cultivated for 24 h in black-walled, clear-bottom 96-well microplates  covered with fibronectin . Cells were grown in a humidified incubator at 37 °C and 5% CO2.For fluorescent plate reader Ca2+ concentration were measured on a multiwell plate reader TECAN Spark 20M using FLIPR 6 Calcium Assay Kit from Molecular Devices  at room temperature (25 °C). The 1000× stock solutions of AML were prepared in DMSO. Briefly, the culture medium was removed and the 100 µL loading buffer was added to the cells together with 100 µL AML solutions of different concentrations in Hanks’ balanced salt solution  containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Then the cells were incubated for 2 h at 37 °C. The cells were washed 2 times with 200 µL HBSS containing AML or (S)-AML (1 nM–5 µM) and 20 mM HEPES. The time courses were recorded in each well. Fluorescence was evoked by 485 nm excitation wavelength and collected in a bottom-read mode at 525 nm. Data was recorded every 2 s . To initiate Ca2+ influx, 50 µL of 400 mM KCl solution in HBSS was injected in each well (speed 100 µL/s) at 10 s time point. To get a fluorescence maximum, 15 of 30 µM Ionomycin  solution in HBSS was injected in each well (speed 100 µL/s) at 50 s time point. The ratios between the KCl induced peak minus basal fluorescence, ΔF(KCl), were taken and expressed as a quotient of the maximal increase induced by ionomycin, ΔF (Ionomycin). The dose response curves of AML were calculated using the software package GraphPad Prism version 8.3.1. All data are presented as mean ± SD in 4 independent experiments. Y = Bottom + (Top-Bottom)/(1 + 10^((LogIC50 − X) × HillSlope)) was used where HillSlope describes the steepness of the curve, Top and Bottom are plateaus in the units of the Y axis.Changes in CaV1.2) cells per well. For the confocal imaging cells were seeded in CELLview™ Slides ; 8000 cells per well or in 35 mm glass bottom dish ; well 14 mm, glass thickness #1.5, 30,000 cells per well. The induction of HEK-293 CaV1.2 cells was done by 24 h incubation in doxycycline solution (0.1 µg/mL). For cellular imaging, cells were incubated at 37 °C with 0.1–2 µM AML prepared in Hanks’ balanced salt solution (HBSS) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, from a 10 mM stock solution prepared in DMSO. For co-staining experiments cell were labeled with CellLight® Golgi-emGFP , MitoTracker® Orange, ER-Tracker® Red, CellLight® Early Endosomes-emGFP (fusion construct of Rab5a and emGFP), CellLight® Late Endosomes-emGFP (fusion construct of Rab7a and emGFP), BacMam 2.0  according to the manufactures instructions. 5 µM SynaptoRed C2®  was used for membrane staining. For the nucleus staining, we used 1 µM TMR-Hoechst and 100 nM 610CP-5-COOH-Hoechst probe [For the wide-field microscopy experiments cells were seeded in µ-24 well plates  or glass bottom 12-well plates  at 25,000 (A7r5 and HL-1) or 12,500  were excited with a 488 nm laser (5% intensity) and detected with a regular PMT in the 510–560 nm range. ER-Tracker® Red was excited with a 561 nm laser and detected with the Leica HyD detector set within the spectral range of 600–650 nm. SynaptoRed C2 (FM 4-64) was exited with a 405  or 561 nm laser and detected with the Leica HyD detector set within the spectral range of 590–650 nm. 610CP-5-COOH-Hoechst probe was excited with 610 nm laser and detected with a regular PMT in the 635–650 nm range. For wide-field imaging we used Lionheart FX automated microscope (Biotek) equipped with dry air objectives 4×, 20× and 40× and sample injection system. 16 fields of view in 3 focusing planes, spanning 6 μm in thickness, were acquired per well. Image stitching and focus stacking was performed using in-built Gene 5 software (Biotek). The final images encompassed 1380 × 950 μm field of view and contained at least 220 and up to 2000 cells.Confocal imaging was performed on a Leica SP8  inverted confocal microscope equipped with an HC PL APO CS2 63x/1.40 Oil objective. All live-cell images shown in the main text were acquired using a 700 Hz bidirectional scanner, and a 92.26 × 92.26-μm field of view (1024 × 1024 pixels and 90 nm pixels), pinhole of 95.6 µm diameter (1 AU), pixel dwell time—325 ns. AML was excited with 405 nm laser (3% intensity) and detected with a regular PMT in the 420–470 nm range. CellLighthttp://svi.nl, accessed on 19 February 2021). All acquired or reconstructed images were processed and visualized using Huygens Essential 20.10 or ImageJ 1.53c software [The images in software . Co-loca


The reaction operates at low catalytic loadings under mild conditions with HFIP as a co-solvent. It works with a variety of substrates, including those bearing internal alkynes. It displays complete 5-exo vs. 6-endo regio-selectivity. In addition, except for the highly congested tBu-substituent, the reaction occurs with high Z vs. E stereo-selectivity, making it synthetically useful and complementary to known catalysts.An original multi-cooperative catalytic approach was developed by combining metal–ligand cooperation and Lewis acid activation. The [(SCS)Pd] An original multi-cooperative catalytic approach was developed by combining metal–ligand cooperation and Lewis acid activation. Thanks to cooperativity, not only the efficiency and selectivity of catalysts can be improved, but the diversity and complexity of the obtainable chemical compounds can also be expanded. In this context, metal–ligand cooperativity (MLC) is clearly prominent.2 Here, a ligand participates directly in substrate activation, in addition to modulating the stereoelectronic properties of the metal center. Complexes of the group 7–9 metals featuring non-innocent pincer ligands, in particular amido and pyridine-based scaffolds, occupy a forefront position in this area.3 Tremendous achievements in hydrogenation and dehydrogenation reactions have been reported with these catalysts.4 Extending the scope of transformations5 and developing the MLC catalytic approach further are currently a major endeavor, along with the diversification of complexes displaying MLC.Major breakthroughs have been made in homogeneous catalysis thanks to cooperative approaches.6 The paucity of MLC behavior reported for Pd and Pt complexes,7 despite the prominent role of these metals in homogeneous catalysis, led us to study complexes of type I  additives. The activity and selectivity of the (SCS) Pd,Pt complexes were thereby spectacularly improved.9 The synergy between MLC and proton shuttling is a very appealing approach to boost catalytic performance. It has been proposed for many hydrogenation/dehydrogenation reactions involving MLC10 and recently unequivocally demonstrated by Hazari et al.11 Its impact in intermolecular hydroamination reactions has also been substantiated.7Our group introduced a few years ago an SCS pincer framework featuring an electron-rich indenediide back-bone.f type I . The elei.e. the formation of carbo- and heterocycles by C–C coupling between an enolizable group, typically a β-dicarbonyl moiety, and an alkyne. As pointed out by Enders et al. in their recent review,12 this is a very powerful transformation and much progress has been achieved recently from both methodological and synthetic viewpoints, but several limitations still need to be addressed. While investigating the ability of the MLC Pd indenediide complex to catalyze Conia-ene cyclizations, we discovered that it is extremely efficient and versatile when associated with a Lewis acid (LA). The combined use of TM catalysts and Lewis acids  has attracted increasing interest and led to impressive developments over the last decade,13 including in the Conia-ene reaction.12,14 Yet, synergistic effects between MLC and LA activation have not been realized so far. As reported hereafter, the association of [(SCS)Pd]2 and Mg(OTf)2 provides a first example of such a dual and multi-cooperative catalytic system. It is a very efficient and useful catalyst for Conia-ene cyclizations, displaying broad substrate scope and high regio as well as stereo-selectivity.Encouraged by these results, we wondered about the possibility of developing new multi-cooperative catalytic approaches based on MLC. Another aim was to expand the scope of SCS Pd,Pt catalysts to more challenging transformations. In this work, we focused on Conia-ene cyclizations, 9 A series of hydrogen-bond donors Pd]2 (5 mol% Pd). The best results were obtained with 4-nitrocatechol. Heating at 120 °C for 16 h, full conversion could be achieved in all cases but 3a (only 30% in this case). The expected cyclized products 1b and 4b were selectively obtained from the β-diketone and β-dinitrile substrates 1a and 4a, whereas 2a led to the formation of a secondary product 2c (30% yield) resulting from double addition of the catechol to the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 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="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>C triple bond,16 besides the targeted carbocycle 2b (47% yield). As for the β-diester 3a, no cyclized product 3b was formed, only the secondary product 3c was obtained.To start with, the carbo-cyclization of the β-dicarbonyl compounds 1a–3a and β-dinitrile 4a bearing an alkyne moiety was studied applying the reaction conditions previously optimized for the cycloisomerization of alkynoic acids .9 A seri17 prompted us to screen a series of fluorinated alcohols as additives. This resulted in noticeable improvement of activity and selectivity . The fact that 4a was readily cyclized is particularly noteworthy since β-dinitriles are usually significantly less reactive than β-dicarbonyl substrates in the Conia-ene reaction.12 It is because the linear geometry of the cyano group is not suited for intramolecular hydrogen-bonding and thus tautomerization is more demanding. Under these milder conditions, the β-keto,ester 2a could also be efficiently cyclized and 2b was formed in high yield without a side product. In turn, the β-diester 3a was unreactive. Due to their weaker acidity, β-diester substrates are notoriously less reactive and more challenging in the Conia-ene reaction.12 The contribution of proton shuttling to the carbocyclization of 1a, 2a and 4a in the presence of HFIP is supported by a strong solvent dependence. While toluene and dichloromethane gave results close to those obtained in chloroform, hydrogen-bond accepting solvents such as THF or DMSO completely inhibited the reaction.The known ability of hexafluoroisopropanol (HFIP) to form hydrogen-bond networks and participate in proton shuttling2 is a competent catalyst for the Conia-ene reaction of β-diketo, β-keto,ester and β-dinitrile substrates, when combined with HFIP as a co-solvent. However, it fails to cyclize the corresponding β-diester under the same conditions. Although the [(SCS)Pt]2 complex proved to be superior for the cyclization of challenging alkynoic acids and amides,8 no improvement was observed in the Conia-ene reaction upon changing Pd for Pt. The Pt complex proved to be in fact less active, with only 40% (vs. 90%) conversion of 2a after 16 h  Pd complex, with the activation of the β-dicarbonyl moiety by a LA.18 Multi-cooperative catalytic systems are very attractive but challenging to achieve. They inherently raise compatibility issues and the different components must be carefully adjusted to act synergistically. In the case of the (SCS) Pd complex, the Lewis acid additive may interact with the electron-rich indenediide motif as well as the sulfur atoms and thereby interfere with the Pd–ligand cooperativity.19 A series of triflate salts were evaluated as LA additives to [(SCS)Pd]2 in the cyclization of the β-keto,ester 2a in 4/1 chloroform/HFIP as solvent. Initial screenings were performed at 60 °C with 1 mol% Pd and 20 mol% LA (15 While Cu(OTf)2 and Sc(OTf)3 had no or negative impact on catalysis, significant improvement of activity was observed with Ca(ii), Mg(ii), Zn(ii) and Yb(iii) salts.20 High conversions were achieved in only one hour (>82% vs. only 25% without a LA), demonstrating the compatibility and synergy between MLC with the [(SCS)Pd]2 complex and electrophilic activation of the β-dicarbonyl moiety with a LA. Taking advantage of the catalytic boost triggered by the LA additive, milder reaction conditions were then used. The temperature was decreased to 25 °C and the LA loading was reduced to 5 mol%. Under these mild conditions, Mg(OTf)2 and Yb(OTf)3 led to the best results . The β-diketo substrate 1a was also readily cyclized under these conditions (>95% after 8 h). In contrast, similar results were obtained with and without Lewis acids in the case of the β-dinitrile 4a (85% conv. is achieved in 16 h at 25 °C). Chelating coordination of the two cyano groups is not possible geometrically, and thus the LA cannot facilitate deprotonation, as in the case of the β-dicarbonyl substrates. Most remarkably, the combination of [(SCS)Pd]2 with the LA allowed for the cyclization of the challenging β-diester 3a as well. Here, Mg(OTf)2 significantly outperformed Yb(OTf)3 (83 vs. 13% conversion of 3a within 30 min). Given the superior efficiency, abundance and biological tolerance of Mg over Yb, catalytic studies were continued with Mg(OTf)2 as the LA.21Thus, the indenediide pincer Pd complex [(SCS)Pd] Table S2.15 Looki mol% LA .15 While2, chloroform/HFIP 4 : 1, 25 °C] are disclosed in 2 is crucial to boost the catalytic activity and makes the Conia-ene reaction work at 25 °C with low Pd loadings. Conversely, in the absence of the Pd complex, no cyclized product  was formed with HFIP alone or with a mixture of Mg(OTf)2 and HFIP. The electron-rich indenediide moiety of the SCS ligand also plays a major role, as substantiated by the noticeable decrease in activity observed when shifting to the corresponding 1-Me-2-indenyl Pd complex.To ascertain the need and synergy of all the catalytic components, a series of control reactions were carried out for the cyclization of the β-keto,ester substrate 2a. The results obtained under conditions deviating from the optimal ones  (6–7% after 24 h), as well as [PdCl2(dppe)] (35%). Even the combination of [PdCl2(dppe)] with a base  performed poorly (78% conversion after 24 h) compared to the [(SCS) Pd] system (>95% in 1.5 h).In addition, much lower conversions were observed with [PdCl]via the formation of indenyl [(SCS-H)PdX] species, several catalytic reactions were in situ analyzed by 31P NMR spectroscopy . When the reaction was carried out in the absence of HFIP, only the dimer [(SCS)Pd]2 was observed along the reaction, indicating that it is the resting state. This behaviour is reminiscent of that observed in the cycloisomerization of alkynoic acids,9 and is consistent with the tight association of the two (SCS)Pd fragments. We decided then to evaluate the behaviour of a pre-catalyst bearing Ph instead of iPr groups on the P atoms. The less electron-donating Ph2P PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 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 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>S sidearms led to weaker association of the (SCS)PhPd fragments.8 Only broad signals were again observed in the presence of HFIP, but when the reaction was carried out in the absence of HFIP, minor signals corresponding to indenyl species were clearly detected by 31P NMR spectroscopy. Although [(SCS)PhPd]3 remains the major species, the observation of indenyl species during the catalytic transformation is consistent with the indenediide ligand backbone acting as a base.Seeking to evidence the non-innocent character of the ligand 2 and Mg(OTf)2 is depicted in double activation of the substrate: the LA chelates the β-dicarbonyl moiety, the acidic proton is transferred to the electron-rich indenediide backbone of the SCS ligand while the alkyne coordinates to palladium (intermediate A); (ii) cyclization: the ensuing enolate adds to the π-activated CC triple bond to give the alkenyl Pd complex B; (iii) product release: back-transfer of the proton from the ligand backbone to the C(sp2) atom bonded to Pd results in proto-depalladation and regenerates the indenediide Pd catalyst. HFIP is presumed to help dissociate the dimeric pre-catalyst,17 and to promote proton shuttling from the substrate to the indenediide moiety in step (i) and from the ligand backbone to the C(sp2) carbon at Pd in step (iii).A simplified 3-step mechanism for the Conia-ene reaction catalyzed by the association of [(SCS)Pd]exo cyclization were investigated first. The (SCS)Pd–Mg association proved to be particularly efficient to cyclize β-diesters 5a–7a featuring an adjacent heteroatom (N or O). The amide-tethered substrate 5a was quantitatively cyclized in only 5 min at 25 °C. Shifting the amide moiety at the exo position (6a) slowed down the transformation somewhat, but it still proceeded rapidly and efficiently at 25 °C (>95% in 50 min). Cyclization of the ether-tethered substrate 7a was more challenging, but heating to 75 °C enabled to achieve high conversion (90%) in only 4.25 h.The scope and functional group tolerance of the reaction were then explored . Our aimExo cyclization of β-keto,esters such as 8a featuring a flexible butylene tether is tricky. It has only been scarcely achieved, using a Au(i) complex featuring a tris-alkynyl phosphine with bulky end caps  or In(NTf2)3 .22,23 Pleasingly, the methylene-cyclohexane product 8b could also be obtained efficiently using the [(SCS)Pd]2 complex (5 mol% Pd) and Mg(OTf)2 (20 mol%) within 20 h at 75 °C (90%). The dual catalytic system proved to be particularly efficient when the enol position bears an oxygen or nitrogen atom. Such substrates have been shown by Hatakeyama et al. to provide straightforward entry to valuable heterocycles and very good results in terms of activity and selectivity were obtained using In(OTf)3 with DBU (5 mol% each) in refluxing toluene.24 In our hands, the β-diester substrates 9a–11a reacted smoothly and high yields (>85%) were achieved with low catalytic loadings . Particularly noteworthy is the formation of the δ-valerolactame 10b which was accomplished in only 2.5 h at 25 °C. Even 7-exo cyclization was possible and highly yielding. Using the same conditions as for 8a, the ε-caprolactame 12b was obtained in 90% yield within 40 h, and the reaction time could be reduced to 7 h using microwave irradiation.25 The preparation of such seven-membered rings was previously described using In(OTf)3/DBU as the catalyst, but in modest yield (41%) and with high catalytic loading (15 mol%).24The length of the linker between the β-dicarbonyl moiety and the terminal alkyne was then modified with the aim to access larger rings (8a–12a). 6-2 and 4.5 mg of Mg(OTf)2. Merging metal–ligand cooperation and Lewis acid activation, as realized with the [(SCS)Pd]2 complex and Mg(OTf)2, thus appears general, practical and of synthetic value. It promotes 5, 6 and 7-exo cyclization of β-dicarbonyl substrates featuring terminal alkynes under mild conditions, and gives access to a variety of carbo- and heterocycles.To assess the robustness of the Pd–Mg dual catalytic system, the cyclization of the amide-tethered substrate 10a was carried out under the optimized conditions  but under open-flask conditions and with technical grade solvents. The δ-valerolactame 10b was obtained in similar yield to under inert conditions, demonstrating that the reaction does not require stringent precautions. Furthermore, when the catalytic loading was reduced by a factor of ten (0.1 mol% Pd and 0.5 mol% Mg), the product was still obtained in very high yield (>95% in 18 h at 50 °C). These reaction conditions were successfully applied to larger scale: 920 mg of pure product 10b were prepared using only 1.4 mg of [(SCS)Pd]exo and endo cyclization products.26 Given the preference of 5-endo against 4-exo-dig cyclization, the β-keto,ester 13a and β-diester 14a were tested first. A longer reaction time was needed for 13a than for 14a (25 vs. 1.5 h) but in both cases, the reaction proceeded readily at 25 °C and afforded high yield (95%) of the 5-endo product. No trace of the less favored 4-exo product was detected by 1H NMR spectroscopy. Our dual catalytic system does not compete with (Ph3P)AuOTf for the formation of cyclopentene 13b (83% yield was achieved in only 5 min at room temperature using 1 mol% Au),27 but on the other hand, the gold catalyst works only with β-keto,esters and failed to cyclize β-diesters.23 As far as the cyclization of 14a is concerned, it had only been efficiently accomplished with Zn halides (10 mol%) but required high temperature and long reaction times .28 We then investigated 5-exo Conia-ene cyclization with internal alkynes (β-diester substrates 15a–19a). The terminal substituent was varied  to assess the impact of sterics and electronics. It should be noted that precedents of Conia-ene cyclization with internal alkynes mostly involve aryl and primary alkyl substituents, while examples with secondary alkyl groups are much rarer and tertiary ones are unprecedented.12,24,27,29 Due to the lower reactivity of these substrates, the catalytic loadings were fixed to 5 mol% Pd and 20 mol% Mg. For the less hindered substrates, with Me (15a) and Et (16a) substituents, cyclization occurred at 25 °C within 3 and 24 h, respectively, while those bearing Cy (17a), Ph (18a) and tBu (19a) groups required heating at 75–120 °C to achieve high conversions. For all substrates, including the tBu-substituted 19a, the reaction was highly selective. The structures of the obtained products were unambiguously determined thanks to thorough 1H and 13C NMR spectroscopic analyses including ADEQUATE, NOESY and dsel-HSQMC-IPAP experiments.15 Complete selectivity in favor of 5-exo vs. 6-endo cyclization was observed, whatever the substituent at the alkyne . For 15a and 16a, a single 5-exo product was obtained, authenticated as the Z isomer. For the substrates 17a and 18a bearing bulkier groups, which required higher reaction temperature, two 5-exo products were formed. In all cases, the Z isomer was very major (Z/E ratio from 7 to 10). In the case of the tBu group, opposite stereo-selectivity was observed. Steric congestion favored here the E product (E/Z of 11.5). Reaction monitoring indicated that it is not due to isomerization of 19b over prolonged heating, but the same E/Z ratio being observed from the beginning of the cyclization. From a mechanistic viewpoint, the Z-selectivity observed with 15a–18a is consistent with the outer-sphere approach outlined in trans to Pd, resulting in the Z product after proto-depalladation.30 This Z-selectivity markedly contrasts with the E-selectivity induced by the In(OTf)3/DBU catalyst .24 In the latter case, the reaction was proposed to proceed by syn carbometalation, with the In(iii) center simultaneously activating the β-dicarbonyl and alkyne moieties.31 Formation of the E product 19b most likely results from isomerization of the vinyl-Pd intermediate B prior to proto-depalladation, due to steric congestion.32The good performance of the dual Pd–Mg catalytic system prompted us to explore then the cyclization of substrates bearing internal alkynes, which are significantly more challenging than terminal alkynes . Being w2 and HFIP, the indenediide-based SCS Pd pincer complex was found to be a very powerful catalyst for Conia-ene cyclizations. It works with a variety of substrates, β-keto,esters as well as β-dinitriles and β-diesters, and gives access with complete regio-selectivity to 5, 6 and 7-membered carbo- as well as heterocycles. It is robust and operates at low catalytic loadings (down to 0.1 mol% Pd and 0.5 mol% Mg). Most remarkably, it is also active and selective with substrates featuring internal alkynes. Whatever the substituent at the terminal position is , 5-exo fully prevails over 6-endo cyclization. Moreover, except for the highly congested tBu product, the reaction occurs with high Z-selectivity. This behavior is in contrast with the E-selectivity observed with In-based catalysts, the other known catalysts for internal alkynes. This is due to the way the C–C bond formation and cyclization proceeds with the dual Pd/Mg catalyst; the Mg-chelated enolate attacks the π-coordinated CC triple bond trans to Pd.When combined with Mg(OTf)These results expand the scope of MLC catalysis in terms of metal  and catalytic transformations . Moreover, this work introduces a new multi-cooperative catalytic approach combining MLC and Lewis acid activation. The MLC/LA synergy parallels and complements that involving MLC and proton shuttling. It also represents a new type of dual activation, in which the TM catalyst displays MLC behavior (towards the enolizable C–H bond in this case) while the LA activates another reactive site of the substrate (the β-dicarbonyl motif). Future studies will seek to explore the generality of such MLC/LA synergy and to exploit it in other catalytic transformations.There are no conflicts to declare.SC-012-D0SC05036A-s001


However, the sluggish half-reaction of water oxidation significantly limits the efficiency of H2O2 generation. Here, a benzylamine oxidation with more favorable thermodynamics is employed as the half-reaction to couple with H2O2 generation in water by using defective zirconium trisulfide (ZrS3) nanobelts as a photocatalyst. The ZrS3 nanobelts with disulfide (S22−) and sulfide anion (S2−) vacancies exhibit an excellent photocatalytic performance for H2O2 generation and simultaneous oxidation of benzylamine to benzonitrile with a high selectivity of >99%. More importantly, the S22− and S2− vacancies can be separately introduced into ZrS3 nanobelts in a controlled manner. The S22− vacancies are further revealed to facilitate the separation of photogenerated charge carriers. The S2− vacancies can significantly improve the electron conduction, hole extraction, and kinetics of benzylamine oxidation. As a result, the use of defective ZrS3 nanobelts yields a high production rate of 78.1 ± 1.5 and 32.0 ± 1.2 μmol h−1 for H2O2 and benzonitrile, respectively, under a simulated sunlight irradiation.Photocatalytic hydrogen peroxide (H 2O2 generation represents a promising approach for artificial photosynthesis. Here, ZrS3 nanobelts with controllable disulfide and sulfide anion vacancies exhibit an excellent photocatalytic H2O2 generation performance together with selective oxidation of benzylamine to benzonitrile.Photocatalytic H As a promising liquid solar fuel generated by artificial photosynthesis, hydrogen peroxide (H2O2) has attracted growing attention because of its high commercial value and low transportation cost3. Substantial efforts have been devoted to the development of effective photocatalysts or photocathodes for the H2O2 generation from water and O26. In the photocatalytic process, the sluggish oxidation of water induced by the photogenerated valence holes is a limiting factor for the production of H2O211. Most previous reports focused on improving the half-reaction of O2 reduction, e. g. by consuming holes with sacrificial agents, such as isopropyl alcohol, benzyl alcohol, and 2-PrOH13. However, the development of an alternative oxidation reaction with accelerated kinetics to produce value-added chemicals was rarely reported. On the other hand, selective oxidation of amines to nitriles with lower oxidation potential than water plays an vital role in both laboratorial and industrial synthetic process since nitriles are the important intermediates during the synthesis of fine chemicals, pharmaceuticals, and agrochemicals21. Intensive research has been carried out to synthesize nitriles from primary amines through dehydrogenation28. However, most of the reactions are conducted in organic solvents under harsh conditions, such as high-temperature, exposure to high-pressure oxygen or air, and presence of oxidants. Photocatalytic reactions have been demonstrated to be an effective approach to synthesize nitriles under mild conditions31, but previous works were seriously limited by the use of noble metals as the co-catalysts to realize the dehydrogenation in organic solvents. Thus, the development of artificial photosynthesis in an aqueous and easy scale-up condition with earth-abundant photocatalysts is highly desirable for the production of nitriles in an economically-viable and environment-friendly way.Artificial photosynthesis, i. e. the conversion of solar energy into chemical energy, is considered as one of the promising approaches to synthesize chemicals with the unlimited energy source and minimized environmental problems3) (ICCD PDF no. 30-1498), a layered n-type transition metal trichalcogenide (TMT), has recently drawn great research interest due to the extraordinary properties arising from its unique disulfide anions (S22−)32. ZrS3 has shown a good optical responsivity of 290 mA W−1 with an in-plane hole and electron mobility at a magnitude of 102 and 103 cm2 V−1 s−1, respectively36. In particular, ZrS3 possesses a bandgap of ~2 eV with a more negative conduction band minimum (CBM) than the H2 evolution potential37, making ZrS3 a promising semiconductor for photocatalytic and photoelectrochemical applications. The previous studies on zirconium nitride have demonstrated that its superior performance for O2 reduction stems from the interaction between Zr sites and oxide species, where the Zr d-orbitals make a strong contribution38. Intriguingly, the conduction band of ZrS3 is mainly composed of Zr d-orbitals35, which has a much more negative potential than the reducing potential of O2 to H2O2. Therefore, ZrS3 shows great potential for the photocatalytic H2O2 generation.Monoclinic zirconium trisulfide  environments in monoclinic ZrS3 lattice, where S1 denotes the sulfide ion (S2−) and S2, S3 are interpreted as the S22−  is usually obtained by vacuum annealing of monoclinic ZrS3 at elevated temperatures. This suggests that ZrS3 can desulfurize into ZrS2 by the post-annealing at a higher temperature under vacuum46. Only one type of Zr (Zr1) and S (S1) environment exists in ZrS2, where the Zr1-S1 bond length is similar to that in ZrS3  plane . This different from the Sx2− (x ≥ 2) ions that are difficult to bond with metals  with rich S2− ions exposed at the edges  has a higher reducibility and can be easily intercalated into host materials. Li can be easily dissolved in solvents like ammonia or ethanediamine to form Li-based complex for the solvothermal treatment, which has been widely utilized to enhance the transition temperature of superconducting materials51 and in particular to induce oxygen vacancies on TiO252. Therefore, such Li-based treatment could be an effective approach to induce S2− vacancies on ZrS3.There are three categories of S  was obtained by the re-annealing of the as-grown ZrS3 NBs at 700 °C for different time  under vacuum. ZrSS2-x with S2− vacancies (ZrS1-yS2-x) was prepared through a low-temperature solvothermal treatment by using Li-dissolved ethanediamine with different amounts of Li . We denote ZrSS2-x NBs annealed for X time as ZrSS2-x(X) and ZrS1-yS2-x NBs annealed for X min and treated with Y mg Li as ZrS1-yS2-x(X/Y). The x-ray diffraction (XRD) pattern indicates the formation of ZrS3 in the monoclinic phase (ICCD PDF no. 30-1498), and the vacuum annealing and further Li treatment did not induce any phase transition in ZrSS2-x(15) and ZrS1-yS2-x(15/100) NBs  NBs, due to the introduction of sulfur vacancies that reduce the crystallinity of ZrS3, as observed in the high-resolution transmission electron microscopy (HRTEM) image  characterization  shows an obvious lattice disorder with relatively poor crystallinity. This suggests that the introduction of sulfur vacancies could lead to a decreased crystallinity in ZrS3, consistent with the XRD results.ZrSers Fig. . Scanninion Fig. . It is d3 and ZrSS2-x (15) NBs absorb light with the wavelength up to ~650 nm, corresponding to a bandgap of 2.02 eV  NBs present a slight red-shift of absorption spectrum, revealing a smaller bandgap of 1.98 eV. The Mott−Schottky plots for all three samples exhibit positive slopes, indicating the n-type behavior of ZrS3  of ZrS3, ZrSS2-x(15), and ZrS1-yS2-x(15/100) are estimated to be −0.10, −0.11, and −0.18 V versus reversible hydrogen electrode (VRHE), respectively , and ZrS1-yS2-x(15/100) can be directly determined by their Efb. Based on the Mott−Schottky , and ZrS1-yS2-x(15/100) were revealed to be −0.10, −0.11, −0.18 VRHE (CBM) and 1.92, 1.91, and 1.80 VRHE (VBM), respectively , and ZrS1-yS2-x(15/100) are higher than the potential for two-electron reduction of O2. Previous studies have shown that the oxidation potential of benzylamine lies higher than that of water, and the benzylamine oxidation was thus used to replace oxygen evolution reaction to couple with photocatalytic and electrocatalytic hydrogen evolution reaction56. This suggests the VBM of defective ZrS3 NBs lying far below the oxidation potential of benzylamine, indicating that these photocatalysts are applicable to the photocatalytic O2 reduction and benzylamine oxidation.As shown in the diffuse reflectance UV–vis spectra Fig. , both ZrrS3 Fig. . These rrS3 Fig. , suggestely Fig. . Efb is tky Fig.  and UV–v3 located at ~ 147, 274, 315, and 524 cm−1 were observed in Fig. grigid), internal out-of-plane vibrations , and S–S diatomic motion (IV: Ags–s), respectively35. The Raman spectra show an obvious red-shift of Ags–s mode by ~5 cm−1 from ZrS3, ZrSS2-x(15), and ZrS1-yS2-x(15/100), originating from the introduction of S22− vacancies40. We also observed a ~3 cm−1 red-shift of Agrigid mode from ZrS3 and ZrSS2-x(15) to ZrS1-yS2-x(15/100). Since the Agrigid is correlated to the vibration of quasi-one-dimensional chains in the direction of c axis  results from the introduction of S2− vacancies, which alters the length of Zr–S bonds within each chain. The similar shift of Agrigid mode was also identified from ZrS3 and ZrSS2-x(15) to the only Li-treated ZrS3 with 100 mg Li (ZrS1-yS(100)) as shown in Supplementary Fig. 3 NBs separately treated by the Li-treatment with different Li amount and by the vacuum annealing for different time. The gradual red-shift of Agrigid mode (difference from 1.6 to 6.1 cm−1) was observed with increasing Li amount, resulting from the increased concentration of S2− vacancies in Li-treated ZrS3 NBs  NBs exhibit a slightly lower binding energy of the Zr 3d core level than ZrS3 NBs, consistent with the results from ZrS3 to ZrS2  shows a significant attenuation of S22− 2p peaks with the nearly unchanged S2− 2p peaks compared to ZrS3 . It is worth noting that no clear peak shift was observed for ZrSS2-x(15), which results from the almost retained electron density around the S sites, as revealed by the Mott-Schotty results. After further Li treatment, both Zr 3d and S 2p core levels of ZrS1-yS2-x(15/100) NBs shifted to the lower binding energy by ~0.3 eV regarding ZrSS2-x(15)  was clearly lower than that of ZrSS2-x(15)  further suggest the separate introduction of S2− vacancies by the Li treatment  Fig. , reveali22− dangling bonds is located at g = 2.007158. Similarly, the characteristic peak of Zr-S2− dangling bonds is located at g = 2.0082  NBs suggests the formation of both S2− and S22− vacancies  than that of ZrSS2-x(15) indicates more sulfur vacancies existing in ZrS1-yS2-x(15/100) NBs60. To have a direct view of the atomic arrangement for ZrSS2-x(15) and ZrS1-yS2-x(15/100) NBs, the high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images were obtained, where the atomic sites can be determined by comparing the HAADF-STEM image with the crystal structure of ZrS3 lattice along the [001] direction  demonstrates the missing atoms only emerging on the S22− sites, as indicated by the red dashed circles in Fig. 2− (indicated by yellow dashed circles) and S22− sites for ZrS1-yS2-x(15/100)  investigation was also carried out to detect the vacancy structure. A characteristic peak can be clearly detected at g = 2.0071 for all the vacuum annealed samples in Supplementary Fig. ies Fig. . The hig00) Fig. .3 NBs for reducing O2 to create the reactive oxygen species (ROS) was first evaluated by the EPR trapping experiment using 5,5-dimethyl-1-pyrroline N-oxide (DMPO). As illustrated in Fig. 2•− were observed for all NBs, confirming the generation of O2•−63. The introduction of S22− vacancies was found to enhance the reduction of O2 to O2•−, and the additional introduction of S2− vacancies led to a further increased photocatalytic activity. The correlation of types and density of sulfur vacancies with photocatalytic activity was further examined by the iodometry12 under the irradiation of AM1.5G simulated sunlight with the presence of benzyl alcohol as the hole scavenger. As shown in Supplementary Figure 1-yS2-x(15/100) NBs with the x = 0.36 and y = 0.13 (16.3% sulfur vacancies) exhibit the best performance for photocatalytic H2O2 generation. When the Li amount and annealing time were simultaneously less than 150 mg and 20 min, respectively, the photocatalytic activity increased with the increase of both S22− and S2− vacancies. When the Li amount reached 150 mg or the annealing time reached 20 min, the photocatalytic activity showed an increase at the early stage and then decreased with the increase of annealing time or Li amount, respectively. This indicates that it is harmful to further improve photocatalytic activity with excessive either S22− or S2− vacancies. This is because excessive sulfur vacancies could act as the recombination centers for photogenerated charge carriers for photogenerated charge carriers. To determine the H2O2 formed rate, the production was analyzed by iodometry  NBs possess a high H2O2 evolution rate of 89.6 ± 1.5 μmol h−1 with good reproducibility  agrees well with its absorption spectrum, revealing that the photocatalytic activity originates from the bandgap excitation of ZrS1-yS2-x(15/100)  produces an AQY of 11.4 and 10.8% for the incident light of 400 and 500 nm respectively and demonstrates a good activity even with the excitation extended to the near-infrared region of ~700 nm. Furthermore, the photocatalyst of ZrS1-yS2-x(15/100) is able to maintain its activity after being recycled for the same reaction with both presence of benzylamine and benzyl alcohol, as presented in Fig. p XPS spectra of ZrS1-yS2-x(15/100) after the repeated photoreaction show a weak peak located at ~168.7 eV  after the photocatalytic measurement.The photocatalytic capability of the defective ZrS00) Fig. . In part1-yS2-x(15/100) for H2O2 generation, we further utilized benzylamine to substitute the hole scavenger. The H2O2 evolution rate of ZrS1-yS2-x(15/100) was decreased to 78.1 ± 1.5 μmol h−1 with the same molar amount of benzylamine as benzyl alcohol, due to the slower oxidation kinetics of benzylamine than that of benzyl alcohol. Simultaneously, the benzylamine was oxidized and converted to benzonitrile at a rate of 32.0 ± 1.2 μmol h−1 with a high selectivity of >99%  photocatalyst shows the rates for decomposition of H2O2 of 0.14 and 0.16 h−1 with the presence of benzyl alcohol and benzylamine, respectively, and the rates for formation of H2O2 of 125 and 113 μmol h−1 with the presence of benzyl alcohol and benzylamine, respectively  and ZrS3 NBs, which produced the H2O2 at a rate of 58.5 ± 1.7 and 30.3 ± 1.3 μmol h−1 with the hole scavenger (entry 3 and 1 in Supplementary Table 2-x(15) and ZrS3 show a decreased H2O2 evolution rate of 48.0 ± 1.2 and 18.1 ± 1.2 μmol h−1 with the use of benzylamine, and the corresponding benzonitrile generation rates are 20.7 ± 1.2 and 7.0 ± 1.0 μmol h−1, respectively , ZrSS2-x(15), and ZrS3 reveals the key role of S22− and S2− vacancies on the O2 reduction and benzylamine oxidation.Based on the high activity of ZrSely Fig. . As a re3 NBs on its photocatalytic performance, the transient open-circuit potential measurements were performed on ZrS3, ZrSS2-x(15), and ZrS1-yS2-x(15/100) NBs to reveal the lifetime of photo-induced charge carriers  was significantly increased to 0.69 s as compared to 0.3 s of ZrS3, while the ZrS1-yS2-x(15/100) exhibits a further enhanced lifetime of 0.82 s, as shown in Fig. 3 also suggests the role of S22− and S2− vacancies on improving the carrier lifetime and dynamics , and ZrS1-yS2-x(15/100) NBs were calculated to be 4.00 × 1018, 5.35 × 1018, and 4.58 × 1019 cm−3, based on the estimated width of the depletion region (wd) under the illumination of 55, 46, and 17 nm, respectively (Supplementary Equation 3 and ZrSS2-x(15) suggests that the significantly enhanced carrier lifetime in ZrSS2-x(15) is attributed to the role of S22− vacancies in reducing electron-hole recombination rather than band bending, in agreement with the previous theoretical calculation41. The significantly reduced wd in ZrS1-yS2-x(15/100) indicates a large electric field strength on the surface of ZrS1-yS2-x(15/100), which can accelerate the extraction of photogenerated holes towards the surface and limit the internal band-to-band recombination. Moreover, the small wd in ZrS1-yS2-x(15/100) results in a large conduction region for the free electrons compared to ZrS3 and ZrSS2-x(15), which is beneficial for the electron transport.To provide a deep insight into the effect of defective structures in ZrS, ZrSS2-x5, and Zrkt) and surface recombination (krec) are discussed in the supporting information. Since the kt/(kt + krec) can evaluate the efficiency of charge carrier transfer between the catalyst and reactant43, the clearly higher kt/(kt + krec) of ZrS1-yS2-x(15/100) than those of ZrSS2-x(15) and ZrS3 indicates that ZrS1-yS2-x(15/100) possesses higher efficiency for benzylamine oxidation  presents a slightly higher kt/(kt + krec) compared to ZrS3, as derived from their similar behaviors of kt and krec  and ZrS3 is mainly ascribed to their similar surface band bending extracted from the Mott-Schottky results  with the larger surface band bending thus shows a significantly decreased krec. The slightly decreased krec of ZrSS2-x(15) compared to ZrS3 orginates from the suppression of surface charge recombination by S22− vacancies. The similar behavior of kt for ZrSS2-x(15) and ZrS3 indicates that the introduction of S22− vacancies have a subtle effect on its catalytic capability for benzylamine oxidation. Furthermore, the large increase of kt for ZrS1-yS2-x(15/100) compared to ZrSS2-x(15) and ZrS3 suggests that the S2− vacancies can act as an additional photocatalytic layer for the benzylamine oxidation . The typical IMPS plots and the generalized reaction schematics are shown in Supplementary Fig. ion Fig. . Furtherrec Fig. . The simion Fig. .1-yS2-x(15/100) NBs with S22− and S2− vacancies for the integration of photocatalytic H2O2 generation with the selective oxidation of benzylamine to benzonitrile in water. More importantly, the unique S22− vacancies and S2− vacancies can be controllably induced in the defective ZrS3 NBs by varying the annealing time and Li amount, which promise a prospective strategy for defect engineering. With the introduction of S22− vacancies, the charge carrier recombination is prominently suppressed, and the surface S2− vacancies are revealed to improve the electron conduction, surface hole extraction, and kinetics of benzylamine oxidation. As a result, the photocatalyst of ZrS1-yS2-x(15/100) exhibits a high generation rate of 78.1 ± 1.5 and 32.0 ± 1.2 μmol h−1 for H2O2 and benzonitrile, respectively. Furthermore, ZrS1-yS2-x(15/100) NBs possesses a photoexcitation up to ~700 nm and delivers a high AQY of 11.4 and 10.8% under the incident light of 400 and 500 nm, respectively.In summary, we have developed an efficient photocatalyst of ZrS3 NBs were synthesized through a typical chemical vapor transport process. 0.96 g S  and 0.91 g Zr  powders were mixed, and 5mg iodine  was added as a transport agent. The mixture was sealed in a quartz ampoule (Φ 6 mm × 200 mm) under the vacuum of 10−3 Pa, which was subsequently placed in the center of a two-zone furnace with a temperature gradient of ca. 15 K/cm from center to edge. The furnace was heated to 650 °C and last for 10 h to produce ZrS3 powder, which has a pure monoclinic crystal structure that is stable at this temperature. The obtained 1.87 g ZrS3 powder was then dispersed in isopropanol  at a concentration of 0.5 mg ml−1 followed by the sonication for 15 min. The dispersion was subsequently centrifuged for 10 min at 1006 xg to remove large aggregates. Finally, about 0.6 g ZrS3 NBs (32% yield) were obtained by the collection from the rest of the dispersion by further centrifugation for 10 min at 16099 xg.The ZrS2 (ICCD PDF no. 11-0679) is usually obtained by vacuum annealing of monoclinic ZrS3 at elevated temperature (820 °C)46. This suggests that ZrS3 can be desulfurized into ZrS2 by the post-annealing at a higher temperature under vacuum. It is implied that such transformation can also be realized by the desulfuration of S22− ions, based on our previous results on TiS3 and crystal structure analysis between ZrS2 and ZrS3 in Fig. 140. As a result, the ZrSS2-x NBs were prepared using the previously reported vacuum annealing method. Specifically, 0.6 g ZrS3 NBs were sealed in the quartz ampule (Φ 6 mm × 10 mm) again, which was then heated to 700 °C and last for different time  to fabricate ZrSS2-x NBs. Besides, a certain amount of Li metal pieces  were added into 30 ml ethanediamine  for continuous magnetic stirring in an Ar-filled glovebox . After the Li was completely dissolved, 0.5 g ZrSS2-x NBs were added into the solution, and the obtained solution was subsequently transferred into a 50 mL Teflon-lined autoclave and sealed immediately. Then, the Teflon-lined autoclave was taken out of the glovebox and kept in an oven at 120 °C for 24 h. After cooling down to room temperature, the mixture was first washed in 0.2 M HCl and then rinsed several times in deionized water and ethanol, where the ZrS1-yS2-x NBs (yield > 96%) was finally obtained.Since hexagonal ZrSa X-ray as the excitation source, EPR (JEOL FA200), tapping-mode AFM , and Raman spectroscopy (Horiba Jobin Yvon Modular Raman Spectrometer) with 514 nm laser excitation were employed to characterize different properties of the defective ZrS3 NBs, e.g. atomic and energy band structure. In particular, the samples for the TEM measurements were suspended in ethanol and supported onto a holey carbon film on a Cu grid.UV-Vis-NIR spectrometer (Hitachi U4100), field emission SEM , TEM , XRD (Bruker D8 Advance), XPS (ESCALAB 250Xi) with Al K2O with 1 mmol benzylamine. After the sonication for a few seconds, the mixed solution was bubbled by oxygen for 30 s. Subsequently, the solution was sealed and irradiated under an AM 1.5G simulated sunlight of 100 mW cm−2 derived from a 300 W xenon lamp fitted with an AM 1.5 filter. At certain time intervals, the solution was filtrated by a 0.22 μm Millipore filter to remove the photocatalyst. The aqueous and organic phase products were then analyzed by the iodometry and gas chromatograph (GC) measurements, respectively.50 mg photocatalyst was dispersed in 30 ml H2O2 was analyzed by the iodometry12. Typically, 50 μL 0.4 M potassium iodide  aqueous solution and 50 μL 0.1 M potassium hydrogen phthalate  aqueous solution were added to 2ml obtained aqueous phase product, which was kept for 0.5 h. The mixed solution was then detected by UV–vis spectroscopy on the basis of absorbance at 350 nm, from which the quantity of generated H2O2 was estimated. In addition, to analyze organic phase product from the benzylamine oxidation, the organic liquid was first extracted using ethyl acetate  and then detected by the GC characterization. FihuThe production of H2O containing 1mmol benzyl alcohol. After sonicating for a few seconds, the mixed solution was bubbled by oxygen for few seconds. Subsequently, the solution was sealed and irradiated under an AM 1.5G simulated sunlight of 100 mW cm−2 derived from a 300 W xenon lamp fitted with an AM 1.5 filter. The amount of H2O2 was analyzed by the iodometry. For the action spectrum analysis, the reactions were performed at 298 K under monochromated light irradiation, with the ΦAQY  determined by the following Eq.  NBs was recovered by centrifugation for 15 min at 16,099g and used for the reaction sequence, with water replaced every 3 h during photoirradiation.The the ZrS3OH containing 50 μL DMPO (Sigma-Aldrich for ESR-spectroscopy). After the sonication, the solution was irradiated by a 300 W xenon lamp with a 420 nm filter for 3 min. The resulted solution was subjected to the analysis by using a JEOL (FA200) ESR Spectrometer.4 mg catalyst was suspended in 500 μL CH−2 . The samples on FTO substrates were firstly prepared by a typical electrophoretic deposition method. For details, 25 ml acetone solution containing 20 mg sample and 40 mg iodine was used as the electrophoresis solution. The experimental setup consists of two pieces of FTO that serve as the anodic and cathodic electrodes, respectively. The FTO substrates were immersed in the above solution in parallel with a distance of 1cm, which were kept in the solution for 5 min at a 10 V bias under the potentiostat control. After being calcined for 2 h in a vacuum oven at 100 °C, a uniform film was firmly coated on the FTO substrates. Samples on FTO substrates were directly used as the working electrode, with a Pt wire and an Ag/AgCl (KCl saturated) electrode as counter and reference electrodes respectively. All the samples were illuminated through the sample side (front-side illumination). The photoelectrochemical performance was recorded in 0.1 M Na2SO4 electrolyte with 0.1 mM benzylamine. Mott-Schottky plots were derived from impedance-potential tests conducted at a frequency of 1 kHz in dark. IMPS spectra were recorded by the Zahner Zennium C-IMPS system.The photoelectrochemical measurements were performed in a three-electrode system with an electrochemical workstation (Zahner Zennium) under an AM 1.5G simulated sunlight of 100 mW cmSupplementary InformationPeer Review File