The presence of DMSO suppresses the Al corrosion, thus enabling the use of this metal as the current collector. An activated carbon‐based supercapacitor is investigated in 1 M LiNO3/10 mM H3PW12O40 in a mixed DMSO/H2O solvent and compared with a POM‐free electrolyte. In the presence of POMs, the device achieves better stability under floating conditions at 1.8 V. At 1 kW kg−1, it delivers a specific energy of 8 Wh kg−1 vs. 4.5 Wh kg−1 delivered from the POM‐free device. The H2 evolution is further shifted by the POMs adsorbed on the activated carbon, which is one reason for the improved stability. The POM‐containing cell demonstrates a mitigated self‐discharge, owing to strong POMs adsorption into the carbon pores.A novel redox electrolyte is proposed based on organo‐aqueous solvent and a polyoxometalate (POM) redox moiety. The presence of dimethyl sulfoxide (DMSO) plays multiple roles in this system. Firstly, it enhances the cathodic electrochemical stability window by shifting the H Present and correct: The presence of dimethyl sulfoxide (DMSO) in water can drastically improve the properties of an electrochemical system: the hydrogen evolution shifts to lower potential due to the strong interaction of DMSO with water. DMSO suppresses the corrosion of aluminum and stabilizes the reduced form of polyoxometalates (POMs). Organo‐aqueous electrolytes based on DMSO, H2O and POMs are thus applied in a supercapacitor device. Depending on the specific application, either high energy or high power is desired. Devices, which can provide high energy are based on battery materials, where faradaic  reactions occur.2 g−1).2 and O2 evolution). Organic electrolytes, on the other hand, have safety and cost concerns, they need to be handled under an Argon atmosphere (due to the strictly anhydrous condition requirements), but they have the advantage to exploit a larger ESW, which turns in higher energy density than the aqueous ones.Another intriguing method is the introduction of a faradaic material as a moiety dissolved in the electrolyte.To date, the reported redox‐active electrolytes are mainly aqueous‐based and various redox species are, so far, applied in redox electrolytes for supercapacitors. Bromine and iodine, highly soluble species in aqueous media, which reacts at the positive electrode, were the first moieties used as redox electrolytes.2 and O2. The restriction of the potential difference is also due to the irreversible redox reaction of Keggin‐type polyoxometalates at low potentials in an aqueous medium. However, it is proved that the presence of an additional organic solvent can stabilize the reduction process of polyoxometalates at low potentials.In the field of supercapacitors, Nuckoswka et al. provided a proof of concept of a dual redox electrolyte, where a polyoxometalate serves as a redox specie at the negative electrode, and hydroquinone is confined as redox‐active moiety at the positive electrode.23 in different solvents: pure water, a mixture of water and 1,4‐dioxane (DO), and a mixture of water and DMSO in volume ratio 1 : 1. The presence of the organic solvent significantly shifts the H2 evolution to lower potentials, thus enlarging the electrochemical stability window (ESW). Hydrogen evolution starts at −1 V vs. Ag/AgCl in the pure water‐based electrolyte, while it starts at −1.7 V in the presence of DMSO. In the electrolyte based on DO‐H2O some decomposition already starts at −1.1 V vs. Ag/AgCl, followed by H2 evolution, which starts at about −1.6 V vs. Ag/AgCl. This effect can be attributed to the modification of the water structure induced by the presence of the second solvent. DMSO causes the breaking of hydrogen bonds between water molecules because the interaction DMSO‐H2O is stronger than the interaction H2O‐H2O.2O.2O will be present (suppression of H2 and O2 evolution).Figure 3PW12O40 heteropolyacid (HPOM) to the 1 M LiNO3‐based electrolytes. In agreement with previous electrochemical studies, HPOM in the pure aqueous system undergoes an irreversible redox reaction at 0.9 V vs. Ag/AgCl, as displayed in Figure 2O. Therefore, DMSO‐H2O mixed solvent was selected for supercapacitor application in next sections. In the experiment of Figure Figure 2O3 layer).2O electrolytes. Consecutive cyclic voltammograms scanned from 0 to 1.3 V vs. Ag/AgCl show, in the pure water‐based electrolyte, a current increase with progressive cycling, a signature of continuous corrosion. This phenomenon is not visible in the presence of DMSO. This result is of high significance because it allows using Aluminum as a current collector for the AC‐based electrodes.One issue of aqueous‐based electrolytes is that they are incompatible with the Aluminum current collector, which is subjected to corrosion  is calculated and reported as a function of the opening potential.Even if Figure 3/H2O‐DMSO+HPOM (blue curve) and 1 M LiNO3/H2O‐DMSO (black curve) at different cut‐off potentials. For the CV of the system with POMs, oxidation and reduction peaks characteristics of fully reversible redox reactions appear at specific potential values where each oxidation peak (O) corresponds to a reduction peak (R). In the curve of the cell with a neat LiNO3 electrolyte, no redox peak appears down to −1.2 V  is absent in neat LiNO3 electrolyte down to −1.2 V vs Ag/Ag+. On the other hand, for the cell with 1 M LiNO3/H2O‐DMSO+HPOM, the reversible redox behavior of the POMs is initially absent , and its effects on the aging of the full cell will be discussed later. POMs are superacids materials, and they have acidic protons available in the structure. The influence of faradaic reactions of POMs is confirmed by a lower efficiency of the system containing 1 M LiNO3/H2O‐DMSO+HPOM with respect to the one without POMs .However, the hydrogen desorption potential (0.29 V) coincides with the potential of O1 peak, which is also located at around 0.29 V vs. Ag/Ag+ Figure b and res+ Figure . HoweverM Figure c. This c−1) and high (100 mV sec.−1) scan rates at the cell voltage 2 V are compared in Figure AC/AC full cells were assembled with a mass ratio between the positive and negative electrodes of 2 : 1, and the cells were evaluated in different potential windows . With this ratio, the potentials of positive and negative electrodes remain inside the electrochemical stability window Figure  up to thDue to the presence of redox peaks in the CV, in this work, the capacity, instead of the capacitance, is calculated and compared. Figure The performance of the two devices is also transposed as Ragone plots, which shows the relation between energy accumulated and power. Figure i. e., EIS and GCPL) are periodically checked to monitor the aging level. This type of experiment is more indicative than to perform long‐term galvanostatic cycling because, in supercapacitors, the degradation reactions are more likely to occur at high potentials than under repeated cycling.+ up to a cell voltage of 2.0 V, meaning that the positive electrode works at almost constant potential during the floating period. On the other hand, the potential of the negative electrode is much lower for 1 M LiNO3/H2O‐DMSO than in 1 M LiNO3/H2O‐DMSO+HPOM at the same cell voltage of 1.8 V. In fact, the negative electrode demonstrates nearly constant potential profile throughout the floating period at 1.8 V. It only starts to show some variation when the cell voltage is increased to 2.0 V. At the voltage of 2.0 V, the negative AC electrode operates below −1.3 V vs. Ag/Ag+ which leads to the severe performance degradationHowever, it is crucial also to understand if these cells can operate for a long time and under stress conditions. Accelerated aging on supercapacitors can be performed under potential floating conditions. In this experiment, the cell is kept at the maximum voltage for a specific time, and the performance  to neutral under the influence of bulk electrolyte pH,3/H2O‐DMSO is due to the hydrogen evolution (water reduction), which is stronger than in the cell with 1 M LiNO3/H2O‐DMSO+HPOM, where an improved self‐discharge is probably due to the strong adsorption of POMs species in carbon pores. The potential decay of negative and positive electrodes in the cell with 1 M LiNO3/H2O‐DMSO+HPOM is only 400 mV and 600 mV, respectively.Self‐discharge of positive and negative electrodes in 1 M LiNO3/H2O‐DMSO than in 1 M LiNO3/H2O‐DMSO+HPOM, indicating a more activation controlled processt1/2 curve, where a more linear trend is observed in the case of 1 M LiNO3/H2O‐DMSO+HPOM, indicating a predominant diffusion‐controlled processThe potential versus log t curve in Figure 32O mixtures in supercapacitor devices. Due to the change of water structure, the hybrid organo‐aqueous system provides significant advantages, which can be relevant, not only for supercapacitors but also for other energy storage systems, like batteries. (1) The hydrogen evolution reaction is shifted to lower potentials, giving the possibility to expand the cathodic electrochemical stability window; (2) the corrosion of Aluminum is suppressed, which enables the use of this metal as a current collector. However, a drawback is that the presence of the organic solvent decreases the ionic conductivity.This work highlights the benefit of using DMSO‐H2O solvents is exploited: the presence of DMSO stabilizes the reduced form of phosphotungstic acid (H3PW12O40), and the redox reaction at low potentials becomes more reversible than in the pure aqueous system.Used together with a redox electrolyte based on POMs, other important properties of mixed DMSO‐H3+10 mM H3PW12O40 in mixed DMSO‐H2O solvent displays improved performance in comparison to the analog POM‐free electrolyte. POMs increase the energy density due to the addition of multi‐steps redox reactions to the double layer charge storage. At the same time, the presence of POMs improves the stability of the cell under floating conditions at 1.8 V. The improved stability is explained in terms of hydrogen evolution. The hydrogen evolution is further shifted to lower potentials by the POM adsorbed on the AC electrode. The POMs modify the hydrogen evolution potential, giving rise to a more stable system. The lower self‐discharge behavior further confirms the strong adsorption of POM moieties in the AC pores in comparison to a POM‐free cell.The AC/AC supercapacitor operated in 1 M LiNOIn this work, only one commercial activated carbon, and only one type of POMs were combined as proof of concept. Further work should be done on the matching of activated carbon with tuned pores and functionalities with various types of polyoxometalates. Moreover, in‐situ techniques, such as in‐situ Electrochemical Quartz Microbalance (eQCM) and in‐situ UV‐VIS spectroscopy can help to further understanding the electrochemical mechanism occurring during charge and discharge at the interface electrode‐electrolyte.2O benefits of a low freezing point, future work will be addressed to the characterization of supercapacitor devices at low temperatures. The exploration of other organic solvent to prepare binary organic‐aqueous electrolyte can be of interest for supercapacitor‐ and battery‐related communities.Finally, the physical properties of the hybrid aqueous/non‐aqueous system inherit the merits from both aqueous  and non‐aqueous  systems. Since the system DMSO‐H3, H3PW12O40, 1,4‐Dioxane (DO) and Dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich.LiNO3PW12O40 was dried at 50 °C under vacuum for 24 hours in order to remove the adsorbed water.HThe electrolytes used in this work are listed below:3 in H2O1 M LiNO3 in H2O: DMSO (1 : 1 v/v)1 M LiNO3 in H2O: DO (1 : 1 v/v)1 M LiNO3+1 mM H3PW12O40 in H2O1 M LiNO3+1 mM H3PW12O40 in H2O: DMSO (1 : 1 v/v)1 M LiNO3+1 mM H3PW12O40 in H2O: DO (1 : 1 v/v)1 M LiNO3+10 mM H3PW12O40 in H2O1 M LiNO3+10 mM H3PW12O40 in H2O: DMSO (1 : 1 v/v)1 M LiNOThe conductivity of the electrolytes was measured with a Mettler Toledo conductometer at 23 °C. The pH was measured with a Mettler Toledo (Five Easy f20) pH meter at 23 °C.2) was used as the working electrode and polished with Al2O3 suspension before each experiment. Pt wire was used as the counter electrode and Ag/AgCl as the reference electrode. For the corrosion experiment, Al foil was used as the working electrode. Before each experiment, the electrolyte was purged with N2 flow.The electrochemical study of the electrolytes was done in a glass cell with Teflon cup. A glassy carbon  as conductive additive with a binder solution in the following way: (i) PVDF binder (Solvay) was firstly dissolved in NMP (Sigma Aldrich), (ii) activated carbon and SuperP were then successively added to the binder solution. The final slurries were coated on Al foil with wet thicknesses of 170, 200, 300 and 350 μm. The variation of the thickness allowed us to select appropriate electrodes for the desired mass ratio. The cast layers were dried first for 2 h at 60 °C and then at 80 °C for 10 h to remove any residual solvent. Circular electrodes (with 12 mm diameter) were punched out from the layer and pressed at 7 tons with a hydraulic laboratory press.A commercial activated carbon (Haycarb PLC), which was extensively characterized in our previous worksSwagelok® type cells have been used for the electrochemical experiments in the full cell. AC electrodes were used as positive and negative electrodes in a mass ratio of 2 : 1. An oxidized silver was used as a reference electrode. Positive and negative electrodes were separated by two glass microfiber filter separators . 450 μl of the electrolyte solution was injected into the cell.Swagelok® type cells were also used for half‐cell configuration to determine the electrochemical stability window and the hydrogen storage ability. In this case, the configuration was the same as in the full cell, but an oversized AC electrode (10 times heavier electrode) was used as the counter electrode.Electrochemical experiments in glass cell configuration−1.– cyclic voltammetry on glassy carbon were recorded with a scan rate of 10 mV s−1.– The corrosion test on the Al foil was conducted at 1 mV sElectrochemical experiments in Swagelok®‐type cells (full cells)– Cyclic voltammetry (CV), Potentiostatic Electrochemical Impedance Spectroscopy (PEIS) and Galvanostatic cycling with potential limitation (GCPL) were performed on full cells sequentially by using the following protocol:−1. GCPL was performed at currents ranging from 0.1 to 10 A g−1. In the specific current calculation, the sum of the mass of positive and negative electrode active materials was considered. The cells were cycled in three different voltage ranges . Potentiostatic electrochemical impedance spectroscopy (PEIS) was performed at the bias cell voltage of 0 V in the frequency range 500 kHz–10 mHz and with a sinus amplitude of 5 mV.CV curves were recorded at 5 and 10 mV s−1.– The aging floating experiment was performed by holding the cell voltage at 1.8 V or 2 V for a total of 300 hours. Every 10 hours of floating, the performance of the cell was evaluated by PEIS, and five galvanostatic cycles at 0.5 A g−1, and the voltage was held at that value for 5 hours. Afterward, the voltage drop  was recorded at the open circuit for 24 h.– Self‐discharge experiment: Before measuring self‐discharge, the cell was charged to 1.8 V with 0.1 A gElectrochemical experiments in Swagelok®‐type cells – Electrochemical stability window (ESW)−1. In detail, starting from the open‐circuit voltage (OCV), the potential was increased in the negative or positive direction with 0.1 V steps until the final potentials of −1.6 V and +1.6 V vs. Ag/Ag+, respectively.– ESW was determined with cyclic voltammetry measurements (CV) performed at 1 mv s−1, and the lower potential limit was step‐wise decreased until −1.6 V vs. Ag/Ag+. The positive potential cut‐off was 0.6 V. Each CV is collected after performing three cycles at a given potential limit in order to ensure proper equilibration of the system.The cyclic voltammograms (CVs) were performed at 2 mV sAll electrochemical tests were conducted on a VMP3 Potentiostat (BioLogic) equipped with EC‐Lab software.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


It is applicable to gram‐scale synthesis without loss of yield or selectivity. The mixed 1,1‐diborylalkene products can be utilized in Suzuki–Miyaura cross‐coupling reactions which take place selectivly at the C–B site. DFT calculations suggest the NaOtBu‐catalyzed mixed 1,1‐diboration of alkynes occurs through deprotonation of the terminal alkyne, stepwise addition of BpinBdan to the terminal carbon followed by protonation with tBuOH. Experimentally observed selective formation of (Z)‐diborylalkenes is supported by our theoretical studies.The NaO tBu has been developed. The reaction proceeds in a regio‐ and stereoselective fashion affording (Z)‐diborylalkenes bearing orthogonal boron protecting groups.John Wiley & Sons, Ltd.Transition‐metal‐free mixed 1,1‐diboration of terminal alkynes with BpinBdan catalyzed by NaO Organoboronic acids and their derivatives have become increasingly of interest due to their widespread application in organic synthesis, materials science, and pharmaceuticals2pin2 reacted with alkenylidene‐type lithium carbenoids to afford 1,1‐diborylalkenes via a boron‐based 1,2‐migration. Alkenylidene‐type lithium carbenoids were formed from 1,1‐dibromoalkenes through Li–Br exchange (Scheme 2pin2.tBu)‐catalyzed 1,1‐diboration of terminal alkynes bearing electron‐withdrawing substituents., 1, 19e], 1e scarce., 9a], , 1y Scheme c.19CyAPDI)CoCH3 as the catalyst . Analysis of the reaction mixture by GC‐MS showed the presence of a trace amount of by‐product, which might be the E‐isomer or 1,2‐isomer, with the same mass and similar fragmentation pattern as 2a. A screen of Brønsted base catalysts revealed that NaOtBu was superior when compared to LiOtBu, KOtBu, and Cs2CO3 (Entries 1–4). Weaker organic bases, such as DABCO or Hünig's base (iPr2EtN), as catalysts were inefficient (Entries 5 and 6). A control reaction (Entry 7) revealed that NaOtBu was essential for this diboration. Further screening of the amount of NaOtBu, , afforded lower yields of 2a (Entries 8–10). Only a trace of product was obtained when 1 equivalent of NaOtBu was used (Entry 11). A survey of solvents revealed that CH3CN was optimal (Entries 12–15). GC‐MS analysis of the crude reaction mixtures showed that 2a was the main product, indicating excellent regio‐ and stereoselectivities .We initially studied the reaction using ethyl propiolate 1 was tested  to much larger tert‐butoxy group (2c) provided the desired products in high yields. Substrates with cyclohexyloxy (2d), benzyloxy (2e), furan‐2‐ylmethoxy (2f), and naphthalen‐2‐ylmethoxy (2g) carbonyl groups, afforded the corresponding products in moderate to high yields (43 %‐78 %). The 1,1‐diboration of phenyl propiolate (1h) and naphthalen‐2‐yl propiolate (1i) gave products in good yields of 65 % and 75 %, respectively. Notably, in the presence of competing internal alkyne (2j) or alkene (2k and 2l) moieties, 1,1‐diboration proceeded at the terminal C≡C bond selectively. Propiolamides 1m and 1n were also compatible with this diboration protocol. Increasing the reaction time to 10 h resulted in increased conversion, and the corresponding products were isolated in 87 % and 50 % yields, respectively. Finally, this method enables a convenient gram‐scale synthesis (5 mmol) without loss of yield, as demonstrated for 1a . The structure and stereochemistry of the 1,1‐diborylalkene products was unambiguously confirmed by single‐crystal X‐ray diffraction studies of 2a, 2e, 2j, and 2m  and 2n .With the optimized reaction conditions in hand, the mixed 1,1‐diboration of a variety of alkynoates ed Table . The mod3 generated via deprotonation of ethyl propiolate (1a) by NaOtBu, the more Lewis‐acidic Bpin boron complexes with the alkyne terminal carbon to form anionic adduct 5 via transition state 4‐ts with a barrier (Δ≠G) of 12.8 kcal/mol. With cleavage of the B–B bond, Bdan then irreversibly migrates to the alkyne terminal carbon to generate the allenylic intermediate 7 via transition state 6‐ts, a process which is exergonic by 22.0 kcal/mol. The energy barrier for this step is 18.0 kcal/mol.To understand the mechanism and the stereoselectivity of the mixed 1,1‐diboration reaction, we performed a detailed DFT investigation, the results of which are shown in Figure 3 followed by 1,2‐migration of Bpin moiety to form 7 is calculated to be unfavorable compared to the above pathway. The relative free energy of 10‐ts for Bpin migration is higher than that of 6‐ts by 2.9 kcal/mol. Thus, the preferred pathway for these two steps is nucleophilic attack at the Bpin moiety by 3 followed by 1,2‐migration of Bdan. The contributions of the two boron atoms to the LUMO of BpinBdan were also calculated and they are very similar (0.254 and 0.235). The higher Lewis‐acidity of Bpin due to its more positive NBO charge than Bdan may be responsible for the preference for the formation of anionic adduct 5 which is thermodynamically more stable than 9. The proton of tBuOH, produced by the deprotonation of ethyl propiolate (1a) in the initial step, transfers to the internal carbon of allenolate 7 and generates the 1,1‐diborylalkene intermediate 12 or 14 stereoselectively, followed by substrate‐assisted dissociation of tBuO– to obtain either product 2a or 2a', respectively. There is a strong driving force of more than 20 kcal/mol for the formation of 2a/2a'  starting from 7. Of both isomers, the experimentally observed product 2a is clearly the thermodynamically favored one, and lies 4.4 kcal/mol below 2a'. In addition, the energy barrier leading to 2a via the transition state 11‐ts, in which the tBuOH attacks from the same side as the Bdan group, is 7.5 kcal/mol lower in energy than that of 13‐ts, leading to 2a', with tBuOH attacking from the same side as Bpin. Therefore, 2a is the main product for both kinetic and thermodynamic reasons, and the acetylide anion 3 generated in this step closes the catalytic cycle. This last step is the stereoselectivity determining step, whereas the rate‐determining step is the Bdan transfer to the alkyne terminal carbon, with the overall activation free energy being 18.9 kcal/mol.Another pathway (blue line) in which the Bdan boron complexes with the acetylide anion 2a, both having higher energy barriers, are shown in Figure 7 via transition state 15‐ts firstly, followed by fast generation of 1,1‐diborylalkene intermediate 12 by transfer of the tert‐butanol proton to nitrogen via 17‐ts. The relative free energy of 15‐ts is higher than that of 11‐ts by 10.7 kcal/mol. Another possible concerted pathway via transition state 18‐ts, has a barrier of 20.4 kcal/mol, and thus is ruled out. So, the favored pathway leading to 2a is the one through 11‐ts.The other two possible pathways to form tBu‐catalyzed mixed 1,1‐diboration of alkynes is shown in Scheme tBu generates acetylide A,nBuLi. Species A reacts with BpinBdan, in which the carbanion attacks the Bpin moiety selectively vs. the less electrophilic Bdan group, to form an sp2‐sp3 alkynyl borate intermediate B., , tBu‐catediate B., 21 Then2a with aryl iodides 27a–e, gave the corresponding (Z)‐alkenylboronates 28 as single isomers in moderate yields (Scheme 28a supports our assignment of the (Z)‐configuration .The synthesis of 1,1‐diborylalkenes bearing two different boryl groups (Bpin and Bdan) is particularly attractive because their differing reactivities allow selective and stepwise Suzuki–Miyaura cross‐couplings., 16, 17,s Scheme . A 2D NOtBu. Diverse 1,1‐diborylacrylates and 1,1‐diborylacrylamides with two different boron protecting groups, which were difficult to prepare previously, were obtained in moderate to high yields with excellent atom‐economy. Our DFT calculations suggest a catalytic cycle of acetylene deprotonation, BpinBdan stepwise addition followed by protonation. Finally, Suzuki–Miyaura cross‐coupling reactions of the products occurred exclusively at the Bpin position affording trisubstituted alkenes.In conclusion, we have developed a simple and highly selective mixed diboration of terminal alkynes with BpinBdan catalyzed by inexpensive and readily available NaOhttps://www.ccdc.cam.ac.uk/services/structures?id=doi:10.1002/ejoc.202000128 1959477 (for 2a), 1969050 (for 2e), 1969051 (for 2j), and 1969052 (for 2m) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from http://www.ccdc.cam.ac.uk/structures.Supporting InformationClick here for additional data file.


Cardiac fibrosis is a key factor to determine the prognosis in patient with myocardial infarction (MI). The aim of this study is to investigate whether the transcriptional factor paired‐related homeobox 2 (Prrx2) regulates Wnt5a gene expression and the role in myocardial fibrosis following MI. The MI surgery was performed by ligation of left anterior descending coronary artery. Cardiac remodelling was assessed by measuring interstitial fibrosis performed with Masson staining. Cell differentiation was examined by analysis the expression of alpha‐smooth muscle actin (α‐SMA). Both Prrx2 and Wnt5a gene expressions were up‐regulated in mice following MI, accompanied with increased mRNA and protein levels of α‐SMA, collagen I and collagen III, compared to mice with sham surgery. Adenovirus‐mediated gene knock down of Prrx2 increased survival rate, alleviated cardiac fibrosis, decreased infarction sizes and improved cardiac functions in mice with MI. Importantly, inhibition of Prrx2 suppressed ischaemia‐induced Wnt5a gene expression and Wnt5a signalling. In cultured cardiac fibroblasts, TGF‐β increased gene expressions of Prrx2 and Wnt5a, and induced cell differentiations, which were abolished by gene silence of either Prrx2 or Wnt5a. Further, overexpression of Prrx2 or Wnt5a mirrored the effects of TGF‐β on cell differentiations of cardiac fibroblasts. Gene silence of Wnt5a also ablated cell differentiations induced by Prrx2 overexpression in cardiac fibroblasts. Mechanically, Prrx2 was able to bind with Wnt5a gene promoter to up‐regulate Wnt5a gene expression. In conclusions, targeting Prrx2‐Wnt5a signalling should be considered to improve cardiac remodelling in patients with ischaemic heart diseases. Wnt signal is relatively silent in adult heart tissue,Paired‐related homeobox genes, including Prrx1 and Prrx2, are members of a subfamily of homeobox genes, which promote transcriptional activation by functional studies.As reported, both Prrx2 and Wnt5a were able to be up‐regulated by TGF‐β in myofibroblast and cancer cell.2An expanded Methods section is included in the 2.1The surgery of MI was operated by ligation of left anterior descending coronary artery (LADCA) as described previously.2.2Echocardiography with standard parasternal and apical views was conducted in the left lateral recumbent position as we described previously.2.3t test between two groups. Chi‐square test was applied to comparisons of survival rates. Statistical analyses were conducted using GraphPad Prism 6.0 or IBM SPSS statistics 20.0. A two‐sided P‐value < .05 was considered significant.All quantitative results are expressed as mean ± SD. The normal distribution of data was tested by the Kolmogorov‐Smirnov test before statistical comparisons, and the normality/equal variance was tested to determine whether ANOVA was appropriate. Multiple comparisons were analysed with a one‐way ANOVA followed by Tukey post hoc tests or Bonferroni post hoc analyses. Comparisons between two groups were analysed by unpaired Student's 33.1−/−Apoe mice by performing the surgery of LADCA ligation. Both mRNA and protein levels of TGF‐β, Prrx2 and Wnt5a were increased in ischaemic hearts at the 30th post‐operative day in the MI model, compared to sham  as shown in Figure The direct interaction between Prrx2 protein and Wnt5a promoter in cultured cardiac fibroblasts was determined by using immunoprecipitation (ChIP) analysis as described previously.3.9As seen in Figure 3.10Knowing that TGF‐β via Prrx2 up‐regulates Wnt5a gene expression in cardiac fibroblasts, we finally tested if the Prrx2‐Wnt5a signalling mediates TGF‐β‐induced cell differentiation of cardiac fibroblasts. Though TGF‐β failed to induce cell differentiation if cardiac fibroblasts were infected with adenovirus expressing Prrx2 shRNA Figure A‐D, over4In the present study, we provided the first evidence that TGF‐β up‐regulates Prrx2 to activate Wnt5a signalling through gene transcription, which induces cell differentiation of cardiac fibroblasts. As a result, this contributes to cardiac remodelling and the delayed recovery of heart functions after MI Figure . This noThe major discovery of this study is that Prrx2 regulates Wnt5a signalling via gene transcriptional manner in cardiac fibroblasts. To the best of our knowledge, this is the first study to identify Prrx2 as a transcriptional factor of Wnt5a, which was supported by the following evidence: (a) In Wnt5a gene promoter, the consensus sequence of Prrx2 is found, which is a putative Prrx2 binding site ‘ACAATTTC’; (b) Analysis of luciferase gene reporter told us that Prrx2 may interact with Wnt5a promoter and activate its transcriptional activity; (c) In cells treated with TGF‐β, ChIP analysis revealed the specific binding of Prrx2 with Wnt5a gene promoter if Prrx2 protein in nuclear lysates was purified by using primary Prrx2 antibody but not control IgG; (d) Importantly, both in vitro and in vivo gene knockdown of Prrx2 down‐regulated Wnt5a mRNA and protein levels, while Prrx2 overexpression enhanced Wnt5a gene expression in cultured cardiac fibroblasts. Although Wnt5a gene is regulated by several transcriptional factors, such as Egr‐1, Twist and NF‐κB,Another discovery of this project is that MI‐induced cardiac fibrosis is Prrx2 dependent. Cardiac fibrosis is an important pathological process contributing to the pathogenesis of cardiac remodelling after MI, which is a transition from an early inflammatory phase to fibrotic granulation and maturation stage of cardiac remodelling.An unanswered question in this study that how TGF‐β up‐regulates Prrx2 gene expression in cardiac fibroblasts. The gene function may be regulated in transcriptional, post‐transcriptional and post‐translational levels.Interestingly, we observed that knockdown of Prrx2 did not affect basal gene expression of Wnt5a in cardiac fibroblasts. We reasoned these possibilities to produce this difference. First, another Prrx2‐induced transcription factor is promoting the increase instead of direct Prrx2 effect. Second, Prrx2 is partially involved in the cardiac fibrosis in resting condition.An issue need to be discussed is why the death occurred at the first 10 days for MI mice. In the MI model alone, 32% of mice died of cardiac rupture and 12% mice died of heart failure. While in the MI model of mice with Prrx2 knockdown, 16% of mice died of cardiac rupture and 8% mice died of heart failure. Based on our investigations, we thought the major cause of death is cardiac rupture and Prrx2 knockdown can prevent cardiac rupture.In summary, the present study proposes a role of Prrx2 in cardiac remodelling in response to ischaemic stress in heart. Specifically, when ischaemia is induced, Prrx2 is activated by TGF‐β to enhance Wnt5a signalling, which in turn induces cell differentiation. The inhibition of either Prrx2 or Wnt5a serves to maintain resting condition of cardiac fibroblasts, which is ultimately not enough for promoting cardiac fibrosis. In perspective, Prrx2 or Wnt5a inhibitor maybe a new drug to treat ischaemia‐related cardiovascular diseases, such as acute MI and stroke.None.WWB performed all experiments and drafted the manuscript. ZYT, TCS, XJJ, PL, WDQ, PS and BW partially performed some experiments. JX, ZL, HYY and ZMM gave a lot of critical suggestions to this project. SXW, CL and TG conceived the idea, designed all experiments, convinced the whole project and revised the manuscript. Click here for additional data file.


The N3 wave is a vestibular evoked neurogenic potential detected in some patients with profound sensorineural hearing loss (PSNHL) during brainstem auditory evoked potential (BAEP) analysis. In 1998, Kato et al. mentioned two electropositive waves preceding N3, which we named p1‐p2, but no further description was given.We sought to demonstrate the reproducibility of these waves and hypothesize on their anatomic origin.We used two cohorts of patients with PSNHL. The first cohort comprised 10 patients with N3, allowing us to establish a new test with adequate electrophysiological conditions headed to detect p1‐p2 waves (PN3EP). The second cohort consisted of two groups: group A comprised 10 patients in whom N3 was not detected; and group B comprised 20 patients presenting N3. PN3EP was performed in both groups, of which 50% had cervical myogenic vestibular evoked potentials (cVEMPs).Only group B presented p1‐p2. The PN3EP facilitated the identification of p1‐p2 over BAEP analysis, and their presence correlated well with cVEMPs.P1‐p2 may be covered due to inadequate BAEP setting conditions, and could be generated in the distal neural path that generates the N3 wave. In 1998 Kato T et al. mentioned two electropositive waves preceding N3 in patients with profound sensorineural hearing loss during brainstem auditory evoked potential (BAEP). We ideate a test, PN3EP, that allows a better characterization of these waves that could be distant potentials of the same vestibular pathway generating N3, acting as an “otolithic BAEP equivalent.” ExploraThere are several vestibular evoked neurogenic potentials (VENPs) not based on reflexes that use the auditory capacity of the otolithic organs to study the vestibular system, including the N3, N5, N6 or P10 waveforms .The patients were recruited between 2009 and 2010 in the laboratory of Clinical Neurophysiology at Joan XXIII Hospital, Tarragona. The study group comprised six women and four men with ages ranging from 2 to 60 years. They contributed 14 ears (eight right ears and six left ears) with PSNHL and N3 present in BAEP. From these 10 patients, three had congenital PSNHL and contributed both ears to the study, seven were sudden PSNHL and only one of these patients contributed both ears to the study. The N3 waves of the selected patients complied with the first three criteria established by Murofushi et al. for the N3 definition  oriented to specifically study the prevalence and morphology of p1 and p2.A second cohort of patients was from a prospective and descriptive research study conducted at Joan XXII Hospital between 2010 and 2013. This cohort comprised two groups of patients with PSNHL. Group A included six women and four men, with ages ranging from 6 months to 63 years. They contributed 11 ears (6 right ears and 5 left) with the absence of N3 in BAEP. From these 10 patients, one had congenital PSNHL and contributed both ears to the study, six were sudden PSNHL and contributed with one ear, and three were PSNHL secondary to an infectious process and contributed with one ear. From the group A patients, five were submitted to cVEMP test. Group B comprised 20 patients, 11 women and nine men, with ages ranging from 2 months to 70 years. They contributed 25 ears (13 right ears and 12 left ears) with the presence of N3 in BAEP. Of these patients, four had congenital PSNHL and contributed both ears, 15 were sudden PSNHL and contributed with one ear and in one case with two ears, and one had PSNHL secondary a traumatic head injury and contributed with one ear to the study. The N3 of the selected patients complied with N3 definition criteria . The system included an analogical/digital converter with a 16‐bit resolution for acquired data. The auditory stimulation was presented through adaptable earphones according to recommendations for the practice of clinical neurophysiology, ISO 9000, IEC 601. Responses were recorded by applying surface Ag/AgCl electrodes of 10 mm diameter impregnated with an electro‐conductive gel  on the scalp. The skin was cleaned with an abrasive gel before attaching the electrodes. The cVEMP recordings were obtained using two different devices. The evoked potential recording system (Synergy) used to record the BAEP, but adapted to record cVEMP, and an evoked recording system , which has a rectifier program included.3.1Brainstem auditory evoked potential was performed locating the electrodes according to the 10:20 system . Two channels were recorded with the active electrodes located over the skin region of the mastoid process and both referenced to the vertex electrode (Cz). A stimulation of 90 dBnHL with rarefaction clicks of 0.1 ms duration was used in healthy ears and the intensity was increased to 105 dBnHL in PSNHL ears. The stimuli repetition rate was 11 Hz and the contralateral white noise was applied with an intensity of 30 dBnHL lower than the ipsilateral stimuli. The band pass was 0.1 Hz–3 kHz. The maximum impedance accepted was 5 KΩ. A total of 1,000 trials were averaged over 15 ms sweep duration with a sensitivity of 500 nV. Reject amplitude artifact interval was 40 µV. In the PSNHL ears, the waves were quantified by calculating an average of 1,000 responses that was repeated three times to ensure reproducibility. The response was calculated by performing a “global” average of all 3,000 responses to allow a better identification of p1 and p2. The recording of the electrode located in the ipsilateral mastoid and referred to the vertex was presented.For the PN3EP, the proposed setting of electrophysiological conditions was applied using the same recording montage as for BAEP, but adding another channel with an active electrode located over the skin region of the seventh cervical vertebra spine (EvC7) and referenced to the vertex (Cz). Stimuli (clicks) were produced by delivering square waves of 0.1 ms duration, with alternating polarity, an intensity of 105 dBnHL and a stimuli repetition rate of 20 Hz. The masking condition of the contralateral ear was applied with white noise of 75 dBnHL. The band pass was broadened to an interval of 0.1 Hz–10 kHz; the maximum impedance accepted was 2 kΩ. An average of 5,000 responses was repeated four times to ensure reproducibility. The global average of 20,000 sweeps were recorded over a 5‐ms sweep with a sensitivity of 200 nV. Reject amplitude artifact interval was 20 µV.The cVEMP electrodes were placed over the sternocleidomastoid muscle (SCM) muscle belly (active), medial clavicle (reference), and sternum (earth). Subjects were stimulated in both ears simultaneously while they performed a maximal SCM contraction in supine position .We proposed to describe the p1 and p2 waves by their latency, amplitude and duration. Latency was measured at the beginning of the rising phase of the wave. The amplitude was measured from the beginning of the ascending phase to the highest peak of the wave; and the duration was measured from the beginning of the ascending phase to the end of the descending phase. The selected description variables were arithmetic mean, standard deviation, and a confidence interval of 95% for mean. The medians were used for ordering descriptions. For the means comparison, the normality of the distribution was checked by Kolmorogov‐Smirnov test and Student’s 55.1n = 11) presented reproducible waves, p1 or p2, preceding N3. Of those, 18% (n = 2) presented both p1 and p2. The polarity of these waves in all cases was interpreted as electropositive. We selected two groups of waves: the first group had five waves with latency intervals ranging from 1.1 to 1.4 ms and were considered the p1 type. The second group had eight waves ranging from 1.7 to 2.2 ms and were considered the p2 type . In one patient, no p1, p2 or N3 was detected during the PN3EP. This patient was not included in the statistical description of p1, p2, and N3 waves. No evoked response was detected in the cVEMP from group A patients, whereas all 10 patients from group B presented p13 and n23 evoked potentials with normal values  are set. This fact prevents the evaluation of the effect of dynamic process of alteration or recuperation of the vestibular system, but does allow us to include patients with congenital PSNHL. The patient excluded from the statistical analysis was a 56‐year‐old woman with sudden left PSNHL who did not express evoked potentials during PN3EP and cVEMP. We attribute this finding to a dynamic process affecting the vestibular system as BAEP was performed several years earlier and she presented an important deterioration in her vestibular function in this period.In conclusion, the two electropositive waves preceding N3 described by Kato T et al. appear in a consistent manner with N3. PN3EP allows us to better identify these waves, which we have named p1 and p2. Their electrophysiological characteristics and relationship with cVEMP appearance lead us to hypothesize that these waves could be distal potentials of the same vestibular pathway generating N3, acting as an “otolithic BAEP equivalent.” Although this is a pilot study, the current investigations will help clinicians to test if the hearing loss is accompanied by vestibular impairment.


Scientific Reports 10.1038/s41598-019-40855-5, published online 12 March 2019Correction to: The original version of this Article contained errors.The authors identified the following errors in the published version of the Article: radiocarbon dates were incorrectly reported, and the carbon inventory data was incorrectly processed. The authors repeated processing of the carbon inventory data, and updated the carbon accumulation rate and carbon budget model. While the correction affects the scale of patterns observed, the pattern itself remains unchanged and as such the original conclusions hold.As a result of these corrections, in the Abstract,“In fact, the net carbon export between 2015 and 2018 was 10% of the wetland’s original carbon stock.”now reads:“In fact, the net carbon export between 2015 and 2018 was 8.1% of the wetland’s original carbon stock.”In the Results, section ‘Carbon Content, Inventory, and Wetland Age’,“Transect B contains a swale wetland of 20–26 cm thickness and is similar to the landward portion of Transect A in its carbon content and age .”now reads:“Transect B contains a swale wetland of 20–26 cm thickness and is similar to the landward portion of Transect A in its carbon content and age .”and in the same section,−2 to 210 kg C m−2 for Transect A and 33 kg to 160 kg C m−2 for Transect B. Most of these carbon inventories fall within the range of previously published soil organic carbon inventories for temperate wetlands (30 kg C m−2 to 120 kg C m−2)44. However, cores A3, A7, and B1 all have greater carbon inventories than previously published inventories, possibly due to the greater age of the wetland unit .”“Carbon inventories for both transects are listed by core in Table 2 and range from 69 kg C mnow reads:−2 to 24 kg C m−2 for Transect A and 11 kg to 20 kg C m−2 for Transect B. These carbon inventories fall outside the range of previously published soil organic carbon inventories for temperate wetlands (30 kg C m−2 to 120 kg C m−2), and are closer to the values reported for temperate forests (5 kg C m−2 to 25 kg C m−2)44.”“Carbon inventories for both transects are listed by core in Table 2 and range from 10 kg C mIn Results, section ‘Carbon Budget Model Parameters’,−2 yr−1) and drops steadily landward to 53 g C m−2 yr−1 in the most landward wetland cell, Cell 1 (see Table 4). Transect B has similar carbon accumulation rates to the landward wetlands of Transect A: 76 g C m−2 yr−1 for Cell 1 and 55 g C m−2 yr−1 for Cell 3. The lowest carbon accumulation rates are almost double the average rate for northern peatlands (29 g C m−2 yr−2), and the highest reported carbon accumulation rate is similar to averages found for temperate wetlands (230 to 320 g C m−2 yr−2) (Mitsch and Gosselink 2015).”“The carbon accumulation rate in Transect A is highest in Cell 9 (263 g C mnow reads:−2 yr−1) and drops steadily landward to 6.9 g C m−2 yr−1 in the most landward wetland cell, Cell 1 (see Table 4). Transect B has similar carbon accumulation rates to the landward wetlands of Transect A: 5.7 g C m−2 yr−1 for Cell 1 and 9.4 g C m−2 yr−1 for Cell 3. The highest carbon accumulation rates are similar to the average rate for northern peatlands (29 g C m−2 yr−2), while the carbon accumulation rates in general are much lower than those found in other temperate wetlands (230 to 320 g C m−2 yr−2) (Mitsch and Gosselink 2015).”“The carbon accumulation rate in Transect A is highest in Cell 9  occurred over six months in the summer of 2017. This rapid loss of carbon is large compared to the current carbon accumulation rates: the remaining wetland at Transect A would need to remain undisturbed for 205 years to replace the carbon lost over that six month period.”now reads:“In total, 12% of the original carbon stock at Transect A was removed over the course of the study period, from June 2015 to February 2018. The majority of this loss of carbon (89%) occurred over six months in the summer of 2017. This rapid loss of carbon is large compared to the current carbon accumulation rates: the remaining wetland at Transect A would need to remain undisturbed for 353 years to replace the carbon lost over that six month period.”And in the same section:2 of wetland loss exported 10% of the original carbon stock.”“Between April 2017 and October 2017, 7.0 mnow reads:2 of wetland erosion exported 11% of the original carbon stock.”“Between April 2017 and October 2017, 7.0 mFinally, in the Conclusion,“10% of the original carbon stock was lost between 2015 and 2018, with most of this loss occurring during a six-month period in 2017.”now reads:“8% of the combined carbon stock of the wetlands in Transect A and B was lost between 2015 and 2018, with most of this loss occurring during a six-month period in 2017.”In addition to these changes in the text, Figure 4 and Figure 5 were corrected in the Article. The original versions of these figures are included below as Figure These errors have now been corrected in the PDF and HTML versions of the Article.The overall conclusions of the Article are unaffected by these corrections.


Intramolecular electron transfer (IET) between a redox‐active organic ligand and a metal in a complex is of fundamental interest and used in a variety of applications. In this work it is demonstrated that secondary coordination sphere motifs can be applied to trigger a radical change in the electronic structure of copper complexes with a redox‐active guanidine ligand through ligand–metal IET. Hence, crown ether functions attached to the ligand allow the manipulation of the degree of IET between the guanidine ligand and the copper atom through metal encapsulation. Sophisticated control: We demonstrate that secondary coordination sphere motifs can be applied to trigger a radical change in the electronic structure of copper complexes with a redox‐active guanidine ligand through ligand–metal intramolecular electron transfer (IET). Crown ether functions attached to the ligand allow manipulation of the degree of IET between the guanidine ligand and the copper atom through metal encapsulation. Coordination compounds with redox‐active organic (or non‐innocent) ligands are attractive for several applications. In catalysis, they could act as an electron reservoir that provides electrons for the activation of substrate bonds.9, 10 The prediction of the electronic structure and the energy barriers for IET in a coordination compound with one or more redox‐active ligands is often difficult, and depends not only on intrinsic properties, but also on the environment .25, 26II and CuI atoms.II and soft co‐ligands that with CuI).(PF6) in 88 % yield. Crystals were grown by diffusion of Et2O into a solution of CH2Cl2. Figure + ion binds to all six oxygen atoms of the crown ether function, and in addition to two of the fluorines from the PF6− counterion. The presence of the K+ ion only slightly varies the bond lengths within the guanidino groups (see Table Before studying copper complexes of L](OTf)2 was obtained in 83 % yield by reaction of L with barium triflate in CH2Cl2 for 18 h at room temperature. The compound was isolated as pale‐yellow powder; unfortunately crystals suitable for an XRD analysis could not be grown.The barium encapsulated compound (PF6) and [Ba@L](OTf)2, all recorded in CH2Cl2, are quite similar . A marginal bathochromic shift of the lowest energy band from 324 nm for L to 326 nm for [K@L](PF6) was observed, whereas a hypsochromic shift to 321 nm was found for [Ba@L](OTf)2. The cyclic voltammograms in CH3CN solution . For [Ba@L]2+, a redox process at E1/2=−0.25 V (Eox=−0.17 V) can be assigned to one‐electron oxidation/reduction . Hence the anodic shifts are ΔE1/2=50 mV for potassium cation encapsulation and ΔE1/2=150 mV for barium dication encapsulation. In difference to previously reported N‐aryl aza‐crown ethers,ortho‐Wurster's crown compounds,3CN or CH2Cl2 solution the bonds between the six oxygen atoms of the ligand and both the K+ and the Ba2+ ion are preserved; an equilibrium between free L and [K@L]+/[Ba@L]2+ was not observed.The UV/vis spectra of , [K@L]PF and + and [Ba@L]2+ are very similar . Therefore one might expect sole electrostatics to result in a difference Δ(ΔE) of 2 between the anodic shifts, when K+ is replaced by Ba2+. The larger experimentally obtained difference of Δ(ΔE)=3 might argue for the presence of both electrostatic and inductive effects.in which AcL and its copper complexes 2. The complex [L{Cu(OAc)2}] was obtained as a grey‐blue solid in 65 % isolated yield  (Cu‐O7) and 1.931(4) Å (Cu‐O9). A second oxygen of one of the acetate groups establishes in addition a weak interaction with the copper atom (2.680(4) Å for Cu‐O8). The second oxygen atom of the other acetate group is further away from the copper atom (3.011(4) Å for Cu−O10). As expected, the imino N=C bond distances of the guanidino groups (N1‐C7/N4‐C12) are elongated upon copper coordination (from 1.282(2)/1.286(2) Å in L to 1.341(7)/1.335(7) Å in [L{Cu(OAc)2}]). On the other hand, the structural data do not argue for ligand oxidation by IET to the copper atom.Scheme d Scheme . CrystalL{Cu(OAc)2}] in CH3CN solution shows a signal for a copper‐centered radical (CuII complex), with a g value of 2.120 and an ACu value of 56 G, being in a typical region for CuII‐guanidine complexes.[2Cl2 solution  is similar to that of the free ligand L. Hence all data indicate that the electronic structure of the complex [L{Cu(OAc)2}] is adequately described as CuII coordinated to a neutral bisguanidine L, both in the solid state and in solution.The EPR spectrum of [L{Cu(OAc)2}](PF6) was obtained in 61 % isolated yield by reaction of [K@L] with Cu(OAc)2. Crystals were obtained by diffusion of n‐pentane into a CH2Cl2 solution. The solid state structure is shown in Figure 1‐coordinated (1.928(2) Å for Cu−O7 and 1.970(2) Å for Cu−O9). One additional oxygen atom (O10) interacts with the copper atom (2.643(2) Å for Cu−O10), while another oxygen (O8) is further away (2.978(2) Å for Cu−O8) and rather interacts with the potassium atom of an adjacent complex unit 2}]. The potassium ion is bound to all six oxygen atoms of the crown ether function, and interacts with two fluorine atoms of the PF6− counterion 2}] and [K@L{Cu(OAc)2}](PF6) also look similar. Hence the data confirm the expectation that the electronic structure of the [L{Cu(OAc)2}] complex does not change significantly upon potassium ion encapsulation; a CuII complex with neutral ligand unit prevails.The UV/vis and EPR spectra Figure  of  in 60 % isolated yield. In this reaction it is important to slowly add CuCl2 to a solution of L, maintaining an excess of L throughout the reaction, since CuCl2 could coordinate not only to the guanidino groups but also to the oxygen atoms of the crown ether.n‐hexane into a THF solution. The solid‐state structure is displayed in Figure 2, CuCl2)] measures 44.2°, being almost perfectly in‐between the angle of 90° for a tetrahedral and the angle of 0° for a square planar coordination mode. The reorganization energy, being an important contribution to the barrier for ligand–metal IET, should decrease as a consequence of this special coordination mode,[L(CuCl2)] in the solid state  is in full agreement with the structure, showing a broad signal at ∼g=2.1 due to a copper‐centered radical. Hence, in the solid state the complex is adequately described as a CuII complex with neutral ligand unit.To favor ligand–metal IET, the hard acetate co‐ligands at the copper atom have to be replaced by softer co‐ligands. Therefore, we reacted ion mode,27, 28L(CuCl2)] recorded in three solvents, differing in their relative permittivity ϵr, are shown. In the solvent with the lowest ϵr value , only one broad signal with a g value of 2.112 is found. The shape of the band and its g value clearly argue for a copper‐centered radical. Hence, in THF the complex [L(CuCl2)] is best described as a CuII complex with a neutral ligand L, similar to the situation in the solid state. When the polarity of the solvent is increased , a small signal near g=2 grows in. In CH3CN, a sharp signal at g=2.003 dominates, that is assigned to an organic ligand. Hence in CH3CN solution, the complex is best described as a CuI complex with an oxidized, radical monocationic ligand unit, ⋅L+. The change in the electronic structure with the solvent polarity could be explained by the charge separation in the CuI complex with ⋅L+ ligand unit, leading to a greater solvent stabilization in polar solvents. Hence, in dependence of the solvent polarity, two different redox isomers ] in CH2Cl2 solution  and E1/2=0.42 V (Eox=0.50 V), that are assigned to two ligand‐centered one‐electron redox processes (redox couples ⋅L+/L0 and L2+/⋅L+), on the basis of the comparison with the cyclic voltammogram of L and the similarity to [AcL(CuCl2)] (Scheme II/CuI). Notably, the first oxidation to [⋅L+(CuCl2)] occurs at a potential very similar to that of free L. This is fully consistent with the EPR measurements showing that the ligand unit L in the complex [L(CuCl2)] is in its neutral, reduced form in CH2Cl2 solution.The cyclic voltammogram of [n Figure  shows tw3CN solution  is observed for [L(CuCl2)] (Eox=−0.24 V) with respect to free L (Eox=−0.35 V). The large shift is fully consistent with the formation of the valence tautomer [⋅L+(CuCl2−)] in CH3CN solution by ligand‐to‐metal IET, rendering the further oxidation  of the ligand unit more difficult. In addition, one recognizes a smaller potential difference between the first and second oxidation process in CH3CN solution compared with the measurements in CH2Cl2 solution. However, a closer inspection shows that the first oxidation and reduction waves consist of two contributions, which separate more clearly in the cyclic voltammograms of [K@L(CuCl2)](PF6) and [Ba@L(CuCl2)](OTf)2, and might be caused by redox‐induced electron transfer (RIET) processes, leading to the presence of two valence tautomers upon one‐electron oxidation (see results obtained previously for [AcL(CuCl2)], Scheme 3CN molecules do not initiate changes in the coordination mode, for example, by substituting the chlorido ligands.We also recorded CV curves in CHn Figure . Here, a Figure Eox=0.11 VL(CuCl2)](PF6) was prepared in 67 % yield by reaction of [K@L](PF6) with CuCl2. Diffusion of n‐pentane into a CH2Cl2 solution afforded crystals suitable for a structural analysis by XRD. Figure L(CuCl2)]+ unit, and also the interactions between these units through Cu‐Cl‐K bridges in the solid state. Despite these interactions, there is no significant change in the Cu−Cl bond lengths (2.249(2)/2.219(2) Å for [L(CuCl2)] and 2.263(1)/2.232(1) Å for [K@L(CuCl2)](PF6)), falling in the typical range of CuII‐bisguanidine complexes.L{Cu(OAc2)}](PF6) the potassium ion does not interact with the fluorine atoms of the PF6− counterion. In the solid state, the cationic units [K@L(CuCl2)]+ interact with each other by Cu−Cl⋅⋅⋅K interactions, leading to polymer chains with K⋅⋅⋅Cl distances of 3.029(1) and 3.399(1) Å. The dihedral angle at the copper atom is slightly larger in [K@L(CuCl2)]+ (53.50°) than in [L(CuCl2)] (44.2°). However, due to the interactions between the [K@L(CuCl2)]+ units, this angle is expected to deviate from the preferred value without these interactions (see discussion below).The complex [K@L(CuCl2)](PF6) in CH2Cl2 is similar to that recorded for [L(CuCl2)]  appear, at E1/2=−0.21 V (Eox=−0.13 V) and E1/2=0.35 V (Eox=0.46 V). The non‐reversible copper reduction occurs at a lower potential of −0.73 V (redox couple CuII/CuI). The cathodic shift of the CuII reduction potential could be explained by the reduced electron‐donor character of L upon K+ encapsulation. Hence, the CV curve is in line with an electronic structure with a reduced [K@L]+ unit and CuII. The CV curve recorded for [K@L(CuCl2)](PF6) in CH3CN solution looks different ] ](PF6) in CH3CN solution. The reversibility of the redox processes again indicates that oxidation is not accompanied by a change of the coordination mode of the copper atom; substitution of chloride by acetonitrile could be excluded on the basis of all experimental results.The cyclic voltammogram of  and [K@L(CuCl2)](PF6) are similar, showing a broad signal due to copper‐centered radical and only a very small one due to ligand‐centered radical . Hence, the adequate description is that of a CuII complex with neutral ligand. In Figure L(CuCl2)] and [K@L(CuCl2)](PF6) in CH3CN solution are compared. Both spectra display two signals. A sharp signal with a g value near 2 is assigned to a radical monocationic ligand unit (⋅L+), being much stronger than in CH2Cl2 solution, and a broad signal with a significantly higher g value assigned to a copper‐centered radical (CuII). However, the ratio of these two signals differs, as estimated by double integration . The contribution from the copper‐centered radical is much larger for [K@L(CuCl2)](PF6) than for [L(CuCl2)]. This means that the K+ encapsulation by the crown ether function has an effect on the electronic structure of the complex, as it leads to a higher preference of the valence tautomer with CuII and neutral ligand. Moreover, this is another evidence for a preserved metal encapsulation in solution, because a loss of metal coordination would increase the ionic strength of the solution thereby favoring the other valence tautomer with CuI and monocationic ligand ](OTf)2 by reacting [Ba@L](OTf)2 with CuCl2 in THF solution. The EPR spectrum of [Ba@L(CuCl2)](OTf)2 in CH3CN solution  is visible. This result demonstrates the possibility to massively change the electronic structure of the copper complex by metal coordination at the secondary coordination sphere. To the best of our knowledge, this is the first authenticated example in which metal encapsulation does not only lead to an anodic shift of the ligand and/or metal potential, but to the change of the oxidation state of a redox‐active metal (copper) by IET to the ligand.Finally, we prepared the complex , leading to the presence of a AcL2+ ligand unit together with CuI upon one‐electron oxidation, starting with a complex with reduced AcL ligand and CuII before oxidation.The CV curve ] and [K@L(CuCl2)]+ are in good agreement with the experimental XRD solid‐state structures (see Table S1 in the Supporting Information). The natural bond orbital analysis  and the two chlorido ligands (ca. 20 %), leaving only 15 % spin density for the ligand unit. Hence, the calculated structures for ϵr=1 could safely be described as CuII complexes with a neutral ligand L, in line with the experimental results of EPR spectroscopy for the solid powder material .All DFT‐calculated structures of [LCuCl] and [K@L(CuCl2)] using COSMO with various relative permittivity values to model the solvent effect, ranging from ϵr=1 to 46.7. At this place we want to stress that one should be cautious with the interpretation of the results of these calculations. For some values of the relative permittivity, different structures were obtained varying only slightly in their energy. Moreover, it is not clear if we found in all cases the lowest‐energy structure. For the plot in Figure ϵr=1, about 15 % of the spin density of [L(CuCl2)] resides on the ligand. This value rises with increasing ϵr value . For lower ϵr values, the electronic structure is best described as CuII with neutral ligand unit (L), and at higher ϵr values as CuI with radial monocationic ligand unit (⋅L+). The plot in Figure ϵr value, motivating that this angle is a suitable indicator for the electronic structure. At ϵr=37.5, already 75 % of the spin density is placed on the ligand unit, and the dihedral angle is close to 90°. In line with conversion from CuII→CuI with increasing solvent polarity, the Cu‐N and Cu‐Cl bond distances increase . Not only the spin density and the dihedral angle, but also characteristic bond parameters within the ligand unit signal conversion from neutral to radical monocationic ligand unit with increasing ϵr value . Hence the N1‐C1/N4‐C2 bond distances (bonds connecting the guanidino groups with the C6 ring) decrease (from 1.404/1.398 Å at ϵr=1 to 1.358 Å at ϵr=37.5), and the imino N=C bond distances within the guanidino groups increase (from 1.321/1.319 Å at ϵr=1 to 1.335/1.336 Å at ϵr=37.5). Moreover, the differences in the C−C bond distances within the C6 ring increase.To investigate the influence of the environment, further calculations were carried out for [LCuCl] using CAcL(CuCl2)] . This result argues for an equilibrium between two redox isomers. Nevertheless, in the calculations a continuous change of the spin density distribution with increasing relative permittivity value in the direction toward a ligand‐based radical was found, similarly to the calculations presented in this work. The distinct signals of organic and metal‐based spin density in the EPR spectra at first glance argues for an equilibrium between two redox isomers. However, further studies are necessary to decide on the question if the spin density gradually changes with the solvent polarity or if an equilibrium exists between two valence tautomers. Anyway, the B3LYP+COSMO results are in good agreement with the experimental data, both with and without inclusion of the solvent effect.In previous calculations on  Figure , two str+ or Ba2+). Both the structures and the spin density contributions, respectively show that the electronic structure of the copper complexes (at ϵr=37.5) is significantly changed by the coordination of potassium or barium ions ]+, rising to not more than 24 % at ϵr=37.5  to a CuII complex with neutral ligand L ] (X=acetate or chloride) of this new ligand L studied. The electronic structure of the copper complex [L(CuCl2)] before metal encapsulation changes with the solvent polarity. In nonpolar solvents (CH2Cl2) and in the solid state, the complex is best described as a CuII complex with neutral ligand unit. By contrast, in polar solvents the electronic structure drastically changes to the redox isomeric CuI complex with radical monocationic ligand, due to ligand–metal intramolecular electron transfer (IET). After encapsulation of K+ or Ba2+ ions into the crown‐ether function, the redox isomer assigned to a CuII complex with neutral ligand unit prevails independent of the solvent polarity. We report here the first example of a drastic change in the electronic structure (in polar solvents) through ligand–metal IET by metal encapsulation into the crown ether function, going far beyond the typically observed anodic shift in the ligand potential. The results show that ligand–metal IET could be triggered by coordination at a remote secondary coordination sphere  should be addressed to the authors.SupplementaryClick here for additional data file.


Previously, we demonstrated the cell-free synthesis of three bacteriophages from their genomes: MS2, ΦX174, T7. In this work, we present the complete synthesis of the phage T4 from its 169-kbp genome in one-pot TXTL reactions. This achievement, for one of the largest coliphages, demonstrates the integration of complex gene regulation, metabolism and self-assembly, and brings the bottom-up synthesis of biological systems to a new level.The bottom-up construction of biological entities from genetic information provides a broad range of opportunities to better understand fundamental processes within living cells, as well as holding great promise for the development of novel biomedical applications. Cell-free transcription–translation (TXTL) systems have become suitable platforms to tackle such topics because they recapitulate the process of gene expression. TXTL systems have advanced to where the  Suchng cells . Cell-frEscherichiacoli TXTL toolbox, for example, has opened new perspectives for the synthesis of biochemical systems through the execution of natural or synthetic gene networks  media in an overnight culture. This culture was diluted 50× into fresh LB media and a single 4–5 h wild-type T4 plaque grown on an LB agar plate with E. coli B cells was cored with a flame-sterilized Pasteur pipette and blown into the liquid culture. The culture was then allowed to incubate for 2 h and then monitored until the onset of superinfection. Superinfection was tested for by adding CHCl3 to a small volume of the infected cells and monitoring for lysis after 5 min. The cells were allowed to incubate for an additional 2 h before deoxyribonucleases (DNase) I (Sigma Aldrich) was added to a final concentration of 5 μg/ml. The solution was shaken at 37°C for an additional 5 min before the volume was then centrifuged at 10 000×g for 10 min at 4°C to pellet the infected cells. The supernatant was discarded and the pellet resuspended in 10 ml cold 1× TM  buffer containing 5 μg/ml DNase I. The concentrated cells were lysed with the addition of 200 µl of CHCl3 and heavy vortexing. The solution was centrifuged at 10 000×g for 10 min at 4°C to remove cellular lysis products. This stock was stored at 4°C over CHCl3 until a fraction was removed for further purification. The removed volume was purified by 5–45% (wt/vol) sucrose gradient and centrifuged at 35 000 rpm for 20 min in a swinging bucket rotor in an ultracentrifuge system. The bands were removed, re-diluted by 3× in 1× TM and re-centrifuged at 25 000 rpm for 1 h to pellet the phage. Each pellet was resuspended using 0.2 ml 1× TM buffer at 4°C overnight.Phages were produced using a single-plaque, multi-cycle technique previously well described in Chen et al. . Briefly2.3E. coli B carpet to ensure that no plaques formed, indicating all phage were disrupted by the process or that there was no bacterial contamination, respectively.Concentrated T4 stock was diluted 10× with Millipore H2O (Milli-Q Advantage A10) before adding an equal volume of saturated Tris: Phenol: Chloroform (ThermoFisher Scientific) solution to disrupt structural phage proteins. The layered solution was gently shaken by hand for 2 min until an emulsion was formed. This was immediately followed by 5 min of centrifugation at 4°C at 13 300 rpm. Due to the large size of the T4 genome, all following DNA handling steps were performed using wide-bore pipette tips to avoid shear damage. The aqueous phase was slowly pipetted off while avoiding disruption of the protein boundary. The pipetted volume was again mixed with an equal volume of Tris: Phenol: Chloroform and the process was repeated for a total of three phenol extractions. A final chloroform back extraction was performed to remove phenol from the genomic solution followed by 5 min of centrifugation at 4°C and 13 000 rpm before removing the aqueous phase to a fresh Eppendorf tube. This volume was then estimated and 2.8 volumes of 95% ethanol (EtOH) (ThermoFisher Scientific) were added for a total EtOH concentration of 70%. This was gently mixed and allowed to sit overnight at −20°C to precipitate DNA in solution. This solution was then centrifuged for 30 min at 13 300 rpm at 4°C to pellet the T4 DNA. The supernatant was discarded and 500 μl of 70% EtOH was added. The pellet was washed by flicking the tube and re-centrifuged at 13 000 rpm at 4°C, discarding the ethanol after. This step was repeated again for 2 total washes. The final pellet was allowed to air dry for 20 min at room temperature before resuspension of the DNA pellet in 100 μl of Millipore water. The final genomic concentration was determined using a NanoDrop One at 260 nm. Following genomic extraction, a fraction of T4 DNA was plated on agar with and without an 2.4E. coli TXTL system  used in this work was described previously in several articles  concentrations were adjusted based on the reaction test made, see text  and 0.025 ml of cell culture dispensed on a 1.5% solid LB-agar plate. Plates were incubated at 37°C for 7 h. The error bars shown in the plots are standard deviations of multiple repeats.The bacteriophage was counted using the standard plaque assay using the 2.6To visualize phages originating from the TXTL system, reactions were plated as above at concentrations high enough to completely lyse all cells on the plate. Phages were recovered from the plates by scraping the top agar from the plates into 2.5 ml 1× TM buffer. This extract was homogenized by vortexing and clarified by high speed centrifugation. The supernatant was then filtered through a 0.45 µm sterile filter, and phages were pelleted in a Ti-50.2 ultracentrifuge rotor at 30 000 rpm, 4°C for 90 min (Beckman Coulter). Pelleted phage were resuspended overnight at 4°C in 1× TM buffer. Phages were adsorbed to and imaged on carbon-coated formvar grids stained with 2% w/v uranyl acetate in a Tecnai Spirit TEM (FEI).The goal of the first experiment was to plot the kinetics of phage synthesis to determine the reaction incubation time and the potential yield of the system. Samples from a cell-free reaction set at the optimum biochemical settings  were plated at 1 h interval. No detectable phages were synthesized during the first 2 h. Most of the phages were synthesized between 2 and 4 h of incubation . Phage y9 PFU/ml several times. It corresponds to about 1.66 × 10−3 phage produced per genome added to the reaction. We did not observe increased T4 synthesis when dNTPs were added to the cell-free reaction, suggesting that DNA replication does not occur. However, one limitation to address quantitatively this question was the high viscosity of the reaction due to the size of the T4 genome.Our next experiment consisted in varying the genome concentration in TXTL reaction. The goal was to probe the production capacity limit of the synthesis reaction, using the optimal reactions settings described above. Genome concentrations above the saturating limit are extraneous and genome concentrations below optimum do not fully exploit the assembly pathways and chemical energy present in the reaction. We observed that at 1 nM and above the reaction was saturated . ThroughIn TXTL, some of the components show correlations to phage yield when their concentrations are varied, such as ion concentration and molecular crowders. We optimized the concentrations of magnesium, potassium, and PEG for T4 synthesis. Potassium glutamate (K-glu) and magnesium glutamate (Mg-glu) are essential ions for transcription and translation reactions and therefore highly important to be set at precise concentrations for T4 TXTL synthesis. Across a respective range of 0–100 mM K-glu and 4–7 mM Mg-glu, optimum concentrations for K-glu were found to be between 40 and 80 mM while there was no statistical difference between concentrations tested using Mg-glu . Outsidein vitro  to test random T4 genome uptake by host cells. No plaque was formed in the 30 repeats. Control 1 and 2 covered all the T4 stocks prepared for this work. There were no observed plaques when the T4 genome was added to a cell-free reaction and incubated for <1 min (control 3) in the 10 performed repeats. When rifampicin (100 μg/ml or 122 µM), an inhibitor of the E. coli core RNA polymerase already tested in TXTL (7 plaque forming unit per milliliter (PFU/ml) was measured (control 6). In each of the final repeats, the equivalent of seven cell-free reactions of 12 µl  was plated at 0 (Control 7) and 12 h (Control 8) incubation. No colonies were observed when plated showing that our cell-free expression system does not contain intact E. coli cells, as discussed previously (9 PFU/ml at best). As an additional verification step, however, we visualized the phage T4 by EM from plaques produced from a TXTL reaction . This observation suggests that there are other thermodynamic modes that allow efficient self-assembly of complex biological systems in in vitro reactions lacking natural templates used for self-organization. The demonstration that MS2, ΦX174, T7 and now T4 can be entirely produced in TXTL reactions also paves the way toward phage engineering for biomedical applications. We expect that the all-E. coli TXTL platform can be employed to create synthetic phages, by recombineering for example, useful for phage therapy or other applications (The objective of this letter is to report the synthesis of the largest biological entity so far achieved in a TXTL reaction. By demonstrating that TXTL systems can process a DNA program larger than 100 kbp encoding for about 100 essential genes, we bring the capabilities of the cell-free technology to a new level. We believe that the total synthesis of a phage of this size and complexity is a significant milestone towards the TXTL execution of a minimal cell genome for self-reproduction of a living entity. It has been estimated by different approaches and groups that the minimal set of genes required by a free-living cell is between 300 and 500 genes . The comE. coli . It is uications .The Office of Naval Research [N00014-13-1-0074]; the Human Frontier Science Program [RGP0037/2015]. Noireaux laboratory receives research funds from Arbor Biosciences, a distributor of myTXTL cell-free protein expression kit.Conflict of interest statement. None declared.


The structures and reactivities of organic phosphinidene chalcogenides have been mainly inferred from trapping or complexation experiments. Phosphinidene chalcogenide derivatives appear to be an elusive family of molecules that have been suggested as reactive intermediates in multiple organophosphorus reactions. The quest to isolate “free” phosphinidene chalcogenides remains a challenge in the field. Here, we present the synthesis, IR, and UV/Vis spectroscopic identification of hitherto elusive phenylphosphinidene oxide and phenylphosphinidene sulfide from the corresponding phosphonic diazide precursors. We isolated these higher congeners of nitroso‐ and thionitrosobenzene in argon matrices at 10 K. The spectral assignments are supported by B3LYP/6–311++G and MP2/cc‐pVTZ computations. There they are! Phenylphosphinidene oxide and phenylphosphinidene sulphide can readily be prepared through photoinduced fragmentation of their corresponding phosphonic diazide precursors. They were identified using IR and UV/Vis spectroscopies in combination with computational methods. The stabilization of R−P=E species can be achieved through coordination to metal centers2) and thiooxophosphoranes (R−PS2) are unstable molecules thought to be generated in the thermolysis of suitable organophosphorus precursors.6, PhPO2), the phosphorus analogue of nitrobenzene, under matrix‐isolation conditions through the reaction of triplet phenylphosphinidene (5) with triplet molecular oxygen (3P‐O2).7, namely (4‐methoxy)phenyl phosphine disulphide (6)8)2,2,2,1) and phenylphosphinidene sulfide Ph−P=S (2) by means of IR and UV/Vis spectroscopy  in an argon matrix at 10 K with N2 as the only IR‐invisible byproduct. The IR spectrum of 3 is characterized by an intense group of signals centered at 2153 cm−1  of matrices containing 3 results in very rapid and complete disappearance of its IR signals. From a number of experiments, we identify a set of IR bands  computations. The strong IR bands at 1185 and 1098 cm−1 are attributed to the P=O stretching and C−H deformation modes. The observed splitting of the vibrational bands is due to different trapping sites in the argon matrix. The measured P=O stretching frequency agrees well with a previously observed IR band at 1203 cm−1 for the stabilized neutral 2,4,6‐tri‐tert‐butylphenylphosphinidine oxide.−1 are thus assigned to the C−H out‐of‐plane vibrational modes of the phenyl ring. Overall, the observed IR vibrational bands match the computed fundamentals of 1 (Table S1) very well.Phosphonic diazides represent potentially useful precursors for the generation of free phosphinidene chalcogenides that can be activated either thermally or photochemically, and we have chosen the UV photolysis of phenylphosphoryl diazide  and two weak transitions at 314 nm (f=0.011) and 474 nm (f=0.004) in good agreement with the experimentally observed UV/Vis spectrum  of matrix‐isolated 4 in solid argon for 30 min at 10 K resulted in the complete disappearance of its IR bands and the appearance of a new set of IR bands. New strong bands appeared at 1185, 1084, 743, 706, 682, and 416 cm−1 upon UV irradiation . The nature of the P=S bond in 1 can also be judged by comparison with the P=S stretching modes in related compounds. Several unstable thiophosphines  have been characterized spectroscopically either in the gas phase or have been isolated in argon matrices.−1,−1.2 (Table S2).Following the route for the synthesis of 4 was also investigated in the UV/Vis spectral region. Under reaction conditions similar to the IR experiments described above, irradiation of an argon matrix containing 4 results in a decrease in intensity of the bands at λ=194 and 220 nm, assigned to 4 and formation of new strong bands at λmax=192 and λmax=337 nm assigned to 2 , 331 nm (f=0.227), and 692 nm (f=0.0008) computed at TD‐B3LYP/6–311++G.The photochemistry of 1 with 2  (1.504 and 1.942 Å at MP2/cc‐pVTZ), thus showing double‐bond character. Wiberg bond indices of 1.74 and 1.54 were computed for the P=O and P=S bonds in 1 and 2, respectively, indicating significant double‐bond character. The P=O length in 1 is comparable to the corresponding values in previously reported compounds with R−P=O ligands as well as with an anionic molybdenum‐bound phosphinidene oxide complex with a P=O bond of 1.514 Å.2 is only slightly shorter than the P=S bond distance of 2.028 Å in the NHC adduct of aryl phosphine sulphide.1 and 2 are 1.819 and 1.820 Å, respectively, at B3LYP/6–311++G (1.824 and 1.822 Å at MP2/cc‐pVTZ) with bond‐dissociation energies (BDE) of 73.4 and 68.5 kcal mol−1 . No significant geometric differences were observed in the phenyl rings. This indicates there is negligible electronic delocalization of the P=E moiety with the phenyl ring (see below). According to our computations, 1 and 2 are planar Cs‐symmetric structures with a 1A′ electronic ground state.Next, we compared the key geometrical and electronic parameters of the optimized structure of 2 Figure  A. The P1 and 2 are +0.33 and +0.07 e at phosphorus, while the oxygen and sulfur atoms have considerable negative charges of −0.34 and −0.33 e, respectively. Sulfur forms weaker and longer bonds than oxygen, owing to the larger sulfur atom with larger and more diffuse orbitals, resulting in poorer orbital overlap with the phosphorus orbitals as compared to the oxygen atom. Indeed, the HOMOs  in 1 and 2 are in‐plane orbitals combining the σ(P−C) orbital, the P lone pair, and an in‐plane p‐orbital of the chalcogen. Carbene‐like features are evident from the finding that the HOMO entails the P lone pair and the LUMO the empty pz orbital on phosphorus; the isolobal analogy to a singlet carbene holds.1 is mainly localized over the phenyl ring, while in 2, the HOMO−1 displays pronounced π‐bonding between the phosphorus and sulfur atoms  is smaller than that of 1 (0.14 eV). The resulting smaller HOMO–LUMO gap correlates well with the UV/Vis absorption maxima of 2, which is red‐shifted for 2 (λmax≈660 nm) compared to 1 (λmax≈460 nm).The NPA atomic charges of s Figure  B,C. Whe1 and 2. The facile generation of 1 and 2 opens the door for further experimental studies on their (photo)reactivity.In summary, we present the generation and isolation of phenylphosphinide oxide and phenylphosphinidene sulfide using a combination of photolysis, matrix‐isolation IR, and UV/Vis spectroscopic methods as well as quantum‐chemical computations. The bis‐azide precursors are easy to prepare and readily undergo thermal or photochemical decomposition to give The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


We employ machine-learning-assisted molecular beam epitaxy to synthesize SrRuO3 films whose quality is sufficiently high to probe their intrinsic transport properties. Experimental observation of the five transport signatures of Weyl fermions—the linear positive magnetoresistance, chiral-anomaly-induced negative magnetoresistance, π phase shift in a quantum oscillation, light cyclotron mass, and high quantum mobility of about 10,000 cm2V−1s−1—combined with first-principles electronic structure calculations establishes SrRuO3 as a magnetic Weyl semimetal. We also clarify the disorder dependence of the transport of the Weyl fermions, which gives a clear guideline for accessing the topologically nontrivial transport phenomena.Magnetic Weyl semimetals have novel transport phenomena related to pairs of Weyl nodes in the band structure. Although the existence of Weyl fermions is expected in various oxides, the evidence of Weyl fermions in oxide materials remains elusive. Here we show direct quantum transport evidence of Weyl fermions in an epitaxial 4 3.Despite various predictions, the evidence of Weyl fermions in oxide materials remains elusive. Here, the authors show evidence of Weyl fermions in quantum transport measurements in an epitaxial ferromagnetic oxide SrRuO Although Weyl fermions have been predicted to exist in various oxides8, evidence for their existence in oxide materials remains elusive11. SrRuO3, a 4d ferromagnetic metal often used as an epitaxial conducting layer in oxide heterostructures15, provides a promising opportunity to seek the existence of Weyl fermions in a magnetic material. State-of-the-art oxide thin film growth technologies, augmented by machine learning techniques, may allow access to such topological matter. Here, we show direct quantum transport evidence of Weyl fermions in an epitaxial ferromagnetic oxide SrRuO3: unsaturated linear positive magnetoresistance (MR)20, chiral-anomaly induced negative MR21, π Berry phase accumulated along cyclotron orbits20, light cyclotron masses24 and high quantum mobility of about 10,000 cm2 V−1 s−127. We employed machine-learning-assisted molecular beam epitaxy (MBE)28 to synthesize SrRuO3 films whose quality is sufficiently high to probe their intrinsic quantum transport properties. We also clarified the disorder dependence of the transport of the Weyl fermions, and provided a brand-new diagram for the Weyl transport, which gives a clear guideline for accessing the topologically nontrivial transport phenomena. Our results establish SrRuO3 as a magnetic Weyl semimetal and topological oxide electronics as a new research field.Weyl fermions in a magnetic material have novel transport phenomena related to pairs of Weyl nodes29, such as chiral-anomaly induced negative MR and high mobility21. Compared with space-inversion-symmetry-breaking Weyl semimetals30, time-reversal-symmetry (TRS)-breaking ones are thought to be more suitable for spintronic applications32. For example, since the distribution of Weyl nodes in magnets is determined by the spin texture1, this distribution is expected to be controlled by the magnetization switching technique34. Recent angle-resolved photoemission spectroscopy (ARPES) studies have found experimental evidence for the electronic structure of magnetic Weyl semimetals Co3Sn2S23 and Co2MnGa4, such as the presence of bulk Weyl points with linear dispersions and surface Fermi arcs. Demonstrating the relevance of Weyl fermions in a magnetic material to spintronic and electronic applications requires information on quantum oscillations, which allows us to characterize transport properties of individual orbits in a magnetic Weyl semimetal. However, systematic and comprehensive measurements of quantum transport, including the quantum oscillations, have been hampered in magnetic Weyl semimetals because of the difficulty in achieving a quantum lifetime long enough to observe them in metallic systems. Since specimens in the form of epitaxial films are advantageous for future device applications of magnetic Weyl semimetals, it is urgently required to prepare single-crystalline thin films35 of magnetic Weyl semimetals whose quality is sufficiently high to probe quantum transport properties.Weyl semimetals, which host Weyl fermions described by the Weyl Hamiltonian, have intriguing and fascinating transport phenomena based on the chiral anomaly and linear band dispersion with spin-momentum locking3, a 4d ferromagnetic material7. SrRuO3 is widely used as an epitaxial conducting layer in oxide electronics and spintronics owing to the unique nature of ferromagnetic metal, compatibility with other perovskite-structured oxides, and chemical stability34. Theoretical studies predicted that the electronic structure of SrRuO3 may include a large number of Weyl nodes caused by the TRS breaking and spin–orbit coupling (SOC)  in it.36 However, a definitive conclusion on the presence of Weyl fermions near the Fermi energy (EF) cannot be drawn from observations of the AHE alone10, because the AHE reported so far for SrRuO3 can be well reproduced using a function composed of both intrinsic (Karplus–Luttinger (KL) mechanism) and extrinsic (side-jump scattering) terms38.Theoretically, the presence of Weyl fermions has been predicted for SrRuOOC) Fig. 7, and su3 film (63-nm thick) epitaxially grown on SrTiO3  unsaturated linear positive MR20 (2) chiral-anomaly induced negative MR21, (3) π Berry phase accumulated along the cyclotron orbits20, (4) light cyclotron mass24, and (5) high quantum mobility27. Although the light cyclotron mass, high mobility, and linear positive MR also exist in semiconductors with parabolic bands41, we confirmed the existence of the Weyl fermions in SrRuO3 by observing all of the five signatures.In this study, we conducted comprehensive high field magnetotransport measurements (see Methods section “Magnetotransport measurements”) including quantum oscillations of resistivity  oscillations), on an extraordinarily high-quality SrRuOiO3 Fig. . Our firρxx at 300 K [ρxx(300 K)] and Tρxx(TT: temperature), is a good measure to gauge the purity of a metallic system, that is, the quality of single-crystalline SrRuO3 thin films (see Methods section “Determination of the RRR in SrRuO3”). In fact, high RRR values are indispensable for exploring intrinsic electronic states. More specifically, RRR values above 40 and 60 have enabled observations of sharp, dispersive quasiparticle peaks near the Fermi level by ARPES42 and quantum oscillations via electrical resistivity43, respectively. To form SrRuO3 with quality exceeding current levels, we employed our recently developed machine-learning-assisted MBE (see Methods section “Machine-learning-assisted MBE”)28.The residual resistivity ratio (RRR), which is defined as the ratio of the longitudinal resistivity ρxx vs. T curve of the SrRuO3 thin film shows a clear kink at the Curie temperature (TC) of ∼152 K .The resistivity 2 K Fig. 12, whileT < TF (20 K)], a semimetallic behavior is seen in the MR (ρxx(B) − ρxx(0 T))/ρxx(0 T)  . In addition, as shown in Fig. ρxy(B) curves below TF are nonlinear, indicating the coexistence of multiple types of carriers (electrons and holes). We note that, below TF, the AHE, which stems from the extrinsic side-jump scattering and intrinsic KL mechanisms in SrRuO3,38 is well suppressed due to the small residual resistivity of the SrRuO3 film with the RRR of 84.3, and thus the ρxy(B) curves below TF are dominated by the ordinary Hall effect (see Methods section “Temperature dependence of the AHE in the SrRuO3 film with the RRR = 84.3”). Below 10 K, where the AHE is negligible, both the ρxy(B) values and the slopes of ρxy(B) change their signs in the high-B region, signaling the possibility of the coexistence of high-mobility electrons with low-mobility holes16. Importantly, both the unsaturated linear positive MR and nonlinear Hall resistivity features start to appear simultaneously when the measurement temperature is decreased to the Fermi liquid range [T < TF (20 K)]. This indicates that the unsaturated linear positive MR stems from the electron- and hole-like Weyl fermions.In this Fermi liquid region  and Tρxx on (001) SrTiO3 substrates in a custom-designed MBE setup equipped with multiple e-beam evaporators for Sr and Ru  gas. O3 gas (3 + 85% O2) was introduced through an alumina nozzle pointed at the substrate. Further information about the MBE setup and preparation of the substrates is described elsewhere60. The surface morphology of our SrRuO3 films is composed of atomically flat terraces and steps, as observed by atomic force microscopy28. Together with Laue fringes in the θ–2θ X-ray diffraction patterns28, this indicates the high crystalline order and large coherent volume of our SrRuO3 films.We grew the high-quality SrRuO3 growth. Therefore, only a few papers have reported SrRuO3 films with RRRs over 50.44 While a conventional trial-and-error approach may be a way to optimize the growth conditions, this is time-consuming as well as costly, and the optimization efficiency largely depends on the skill and experience of individual researchers. To avoid such a time-consuming approach and reduce experimental time and cost, we employed machine-learning-assisted MBE, which we developed in previous research28. Here, three important growth parameters  direction of the SrTiO3 substrate  and of the orbital MR (∝B2) provide only a very limited contribution to the net MR response and that the WAL and the orbital MR are negligibly small above 0.5 T. In addition, the contribution of the AMR above 0.5 T is also negligible since the magnetization of the SrRuO3 films saturates at about 0.5 T 39. In our case, however, the Hall resistivity shows nonlinear behavior reflecting its semi-metallic feature −ρxx(0 T))/ρxx(0 T) in the SrRuO3 film, especially in the low-magnetic-field range (0 T < B < 4 T), where the nonlinearity of the Hall resistivity 39 than that of Bi2Se3 (~1%) with the large sheet carrier density of 1.7 × 1016 cm−2.68 In contrast, since the SrRuO3 film with the RRR of 84.3 is metallic, the Hall resistivity (=0.36 μΩ cm) at 2 K and 14 T is more than ten times smaller than the longitudinal resistivity (=3.79 μΩ cm) at 2 K and 14 T. This means that the longitudinal voltage in SrRuO3 is less affected by the Hall voltage.The admixture of a component of the Hall resistivity to the longitudinal resistivity is also ruled out by taking the small Hall resistivity of SrRuO3 film with the RRR of 84.3.Therefore, we concluded that the observed unsaturated linear positive MR above 0.5 T originates from the linear energy dispersion around the Weyl nodes and that the contribution from the conventional bands on the MR is negligible in the SrRuO3 is caused by an extrinsic factor (side-jump scattering) in addition to an intrinsic factor (KL mechanism)38. To determine the contributions of the intrinsic and extrinsic factors to the AHE, we investigated the temperature-dependent scaling of the AHE in the SrRuO3 film with the RRR = 84.3.It is known that the AHE in SrRuOρxy(B) in SrRuO3 is described as the summation of the ordinary 38M⊥: cρsM⊥. Here, the proportional coefficient ρs differs depending on the origin of the AHE, which can be of intrinsic (KL mechanism) and extrinsic (side jump scattering) origin38. As shown in Supplementary Fig. TF (~20 K), clear AHE, which is proportional to the magnetization hysteresis curve , equivalently with a very small residual ρxx, AHE becomes negligibly small at low temperatures. Therefore, ρxy(B) curves below TF in our data are dominated by the ordinary Hall effect.Supplementary Figure 3 film with the RRR of 84.3. As described in the Methods section “Excluding other possible origins of the positive MR in SrRuO3”, the contribution from the trivial bands to the MR, such as orbital MR64, AMR, and WAL65, are negligible above 0.5 T.Here, we exclude other possible mechanisms as the origin of the anisotropic linear negative MR at 2 K for the SrRuO73 is also excluded as an origin of the linear negative MR  and F2 (=44 T) orbits), estimated from the SdH oscillation in Fig. vF is Fermi velocity, kB is Boltzmann’s constant, and TD is the Dingle temperature). The mean free paths of other orbits (F3–F6) are smaller than these two orbits (F1 and F2). Since the mean free paths of the Weyl fermions are larger than the thickness of the SrRuO3 films ; kF, Fermi wave number) is smaller than the film thickness  are not long enough to travel a full cyclotron trajectory at the measurement temperature (=2 K), where only the F1 and F2 SdH oscillation peaks are detected; as shown in Fig. F3–F6 are observed at 0.1 K. Therefore, the contribution of the boundary scattering to the negative MR  dispersion should show the π phase shiftμc is located in the mid-gap between the Landau levels  pinning. In the SdH oscillation, the minimal conductivity is obtained when the chemical potential Nth Landau levels of the quadratic energy band 78m is an effective mass, v is the velocity of electrons in the linear band, and N is the Landau level index. The conductivity becomes maximum at μc, since the density at μc becomes maximum when μc is located at the center of the Landau level  phase shifts, respectively, even when a large number of carriers pin the chemical potential. Therefore, whether the chemical potential is pinned or not in our SrRuO3 film, we can estimate the phase shift by using the LK theory  direction of the SrTiO3 substrate. As we explained in the main paper, the ρxy(B) curves of the SrRuO3 film with the RRR of 84.3 at 2 K is nonlinear, indicating the coexistence of multiple types of the Weyl fermions from which the unsaturated linear positive MR stems. Notably, as shown in Supplementary Fig. dρxy/dB changes its sign from negative to positive with decreasing RRR. In the SrRuO3 film with the RRR of 8.93, clear AHE is observed near zero magnetic field due to its large residual resistivity of 20.2 μΩ cm, and the ρxy(B) curve shows the linear dependence on B above 5 T as highlighted in Supplementary Fig. 3 film with the RRR of 8.93, which are estimated from the slope of the ρxy(B) above 5 T, are 4.04 × 1022 cm−3 and 7.65 cm2 V−1 s−1, respectively. The carrier concentration and the mobility are consistent with the reported values for the trivial Ru 4d bands crossing the EF in SrRuO386. These results mean that the Weyl fermions become more dominant in the transport properties with increasing RRR and that the contribution of the Weyl fermions on the Hall resistivity is negligibly small when the RRR is 8.93.Supplementary Figure TF, indicating again that the nonlinear ρxy(B) is a hallmark of the existence of the Weyl fermions in SrRuO3 and that the Weyl fermions become dominant in the transport when scatterings from impurities and phonons are sufficiently suppressed.As described in the main text, the unsaturated linear positive MR also becomes more prominent with increasing RRR and decreasing temperature below 87 for the exchange correlation functional in the projector-augmented plane wave (PAW) formalism88 as implemented in the Vienna ab-initio Simulation package89. The energy cutoff was set to 500 eV, the Brillouin zone was sampled by an 8 × 8 × 6 Monkhorst–Pack mesh90, and the convergence criterion for the electronic density was defined as 10−8 eV. The effect of electronic correlations in the Ru d shell (4d4 for Ru4+) was taken into account by using the rotationally invariant GGA + U scheme91 with U = 2.6 eV and J = 0.6 eV. The choice of parameters is justified by early estimations92 and is in agreement with other studies94.Electronic structure calculations were performed within the density functional theory and generalized gradient approximation 3 has the Pbnm (#62) space group, which corresponds to the D2h point group with symmetries of inversion (I), three mirror planes , and 180° rotations around the orthorhombic axes . The crystal structure parameters considered in the present study are a = 5.5670 Å, b = 5.5304 Å, c = 7.8446 Å,12 and the atomic Wyckoff positions in fractional coordinates are given as 4c  for Sr, 4b  for Ru, and 8d  for O.The orthorhombic phase of SrRuOd states hybridized with the O 2p states and the electronic spectrum reveals half-metallicity . From Supplementary Tables z61–4 (8–16 meV), which are located near the Y–T line in the Z–Γ–Y–T plane as shown in Fig. µ1 = 8.5 meV and µ2 = 8.8 meV for the F1 and F2 orbitals, respectively).We have selected two pairs of bands I and II for the cases when the magnetization is along the orthorhombic entclass1pt{minima94, there is no clear consensus on whether the electronic spectrum of orthorhombic SrRuO3 in the ferromagnetic state is half-metallic or not, and the result turns out to depend on the details of electronic structure calculations. While our theoretical results are in good agreement with the present experiments, we do not rule out the possibility of a non-half-metallic behavior with both spin channels crossing close to the Fermi level. Assuming that the spin-up states can also lie at the Fermi level, there will be an extra set of band crossings in addition to the Weyl points already defined in a half-metallic scenario.From the obtained results, one can clearly see the presence of the Weyl fermions below and above the Fermi level coexisting with trivial half-metallic bands. However, it is worth commenting on another scenario. According to previous theoretical studies3 substrate breaks the orthorhombic D2h symmetry. The reported crystal structure parameters of epitaxial SrRuO3 on SrTiO3 are a = 5.5290 Å, b = 5.5770 Å, c = 7.8100 Å, 12 According to our electronic structure calculations within GGA + U with U = 2.6 eV and J = 0.6 eV for the monoclinic SrRuO3 on SrTiO3 (Supplementary Fig. D2h symmetry, while additional band crossings may occur due to the lowered crystal symmetry.Finally, it is worth noting that a small monoclinic distortion induced by the SrTiOSupplementary InformationPeer Review File