The achievement of perfect light absorption in ultrathin semiconductor materials is not only a long‐standing goal, but also a critical challenge for solar energy applications, and thus requires a redesigned strategy. Here, a general strategy is demonstrated both theoretically and experimentally to create a planar metasurface absorber comprising a 1D ultrathin planar semiconductor film , a transparent spacer, and a metallic back reflector. Guided by derived formulisms, a new type of macroscopic planar metasurface absorber is experimentally demonstrated with light near‐perfectly and exclusively absorbed by the ultrathin semiconductor film. To demonstrate the power and simplicity of this strategy, a prototype of a planar metasurface solar cell is experimentally demonstrated. Furthermore, the device model predicts that a colored planar metasurface perovskite solar cell can maintain 75% of the efficiency of its black counterpart despite the use of a perovskite film that is one order of magnitude thinner. The displayed cell colors have high purities comparable to those of state‐of‐the‐art color filters, and are insensitive to viewing angles up to 60°. The general theoretical framework in conjunction with experimental demonstrations lays the foundation for designing miniaturized, planar, and multifunctional solar cells and optoelectronic devices. They hold great promise for energy‐efficient buildings because they can be integrated with both interiors and exteriors, such as facades, windows, and offices of the buildings. So far, two main approaches have been used to improve perovskite solar cell aesthetic. The first is to employ thick opaque perovskite layers  with reflected colors generated from the nanophotonic structures incorporated on the top or at the bottom of the cells.22.12.1.1n1, n2, κ2, d2, n3, d3, n4, and κ4) are involved in designing the planar metasurface absorber. What is lacking is a general theoretical framework to design this absorber in a more direct and quantitative way. The well‐known Gires–Tournois interferometers used in previous studiesAs shown in Figure E(z) is the electric field in the ultrathin semiconductor film, E0 is the incident electric field, and λ is the wavelength. Zero reflection givesIn the first step, we investigated the dissipative properties of planar metasurfaces. We have derived the expression to calculate the absorption rate per unit length (absorbed energy at a given depth) for multilayer structures based on the transfer matrix theory in our previous works.d3 > 0, we assume thatSince the semiconductor film is much thinner than the wavelength and the electric field can penetrate into the transparent material spacer for Aexc = 1), we getThis assumption is different from that in Park et al.12 is the reflection phase shift at the interface between medium prop is the propagation phase shift in the semiconductor film, and ψ234 is the reflection phase shift at the interface between the semiconductor film and the transparent spacer (2–3 interface). ψ12 is equal to the phase angle of r12, the reflection coefficient at the 1–2 interface, expressed aspm = pn + piκ is the complex refractive index of layer p. ψ234 is equal to the phase angle of r234, the reflection coefficient at the 2–3 interface, expressed aspqr = (pm − qm)/(pm + qm) and β3 = 2πn3d3/λ. ψprop is expressed asIn the next step, we studied the reflection phase shift behavior at the interface between the semiconductor film and the transparent spacer similar to previous studies.r234) is the imaginary component of r234 and C is a negative real number. Substituting Equations Two assumptions, (i) the magnitude of the complex refractive index of the semiconductor material is much larger than one which is reasonable for most semiconductors and (ii) the metallic substrate is approximated as perfect electric conductor, were applied to get234 can always be designed to be larger than π, that is, Im(r234) < 0, by tuning the transparent spacer thickness, d3, so that we can find the semiconductor film thickness, d2, that is smaller than a quarter wavelength, circumventing the quarter‐wavelength lower limit, as well as satisfies Equation d2, can be designed by Equation d3Equation d3 = 0. This gives ψ234 = π. Substituting this result into Equation d2 = λ/(4n2) corresponding to the quarter‐wavelength thickness limit. d3 = 0 also gives E(z = d2) = 0 because the electric field cannot penetrate into the perfect electric conductor substrate; the resultant ∫0d2|E(z)|2dz is approximated to be equal to |E0|2/2. Perfect absorption leads to κ2 =λn1/(2πn2d2). For the quarter‐wavelength thickness limit, we have κ2 = 2n1/π. Therefore, perfect absorption can be achieved in the ultrathin semiconductor film only for spectra where the extinction coefficient of the semiconductor material is larger than 2n1/π. Otherwise, although the planar metasurface absorber can resonate at the target wavelength determined by Equation Overall, by scrutinizing both the dissipative and optical cavity behaviors of the planar metasurface, we introduce Equations 2.1.22O3) for the transparent spacer, air for medium Figure2O3, and Ag were measured using the ellipsometry method  and shown in Figure 2O3 thickness was then designed to be 20 nm according to Equation 2O3/Ag sample was fabricated using the electron beam evaporation method, and characterized using the scanning electron microscopy shown in Figure 2O3 and Al2O3–Ag interfaces were characterized using the X‐ray photoelectron spectroscopy shown in Figure πn2κ2d2/(λn1) and (4πn2d2/λ + ψ234 − ψ12)/π are both nearly equal to one corresponding well to Equations Now we experimentally prove the concept. Without losing generality, germanium (Ge) was used for the semiconductor film with alumina AlO3 for thFigure2O3 layers are much smaller than the wavelength and Ge refractive index is as larger as 4.35, both the propagation and reflection phase shifts, and thus, the resonant wavelength, change slightly with incident angles. More detailed analysis can be referred to our previous work.Furthermore, the planar metasurface exhibit omnidirectional resonant behaviors, which is also an important feature for solar PV applications. We measured and calculated the spectral reflectivity of the sample for unpolarized light with incident angles up to 60° as shown in 2.22.2.1Figure2O5) for the hole transport layer, a‐Si for the active layer, indene‐C60 bisadduct (ICBA) for the electron transport layer, and an opaque Ag film for the cathode.The planar metasurface structure shown in Figure 2O5 can absorb light at the resonant wavelength. Results also show that the calculated 4πn4κ4d4/(λn1) and (4πn4d4/λ + ψ456 − ψ34)/π are both close to one corresponding well to our theoretical framework.Without losing generality, the resonant wavelength was selected to be 470 nm to produce yellowish appearance as shown in Figure −2, an open‐circuit voltage of 0.6 V, and a fill factor of 0.2, yielding an efficiency of 0.6%. This efficiency is reasonable for a prototype considering the state‐of‐the‐art 10.2% efficiency for a single‐junction cell using a 250 nm intrinsic a‐Si film,3 layer to reduce contact resistance, or by tuning layer thicknesses to optimize electrical properties.The electrical performance of the ultrathin a‐Si solar cell was charaterized by current density–voltage measurements as shown in Figure 2.2.23NH3PbI3−xClx) was used for the active layer as shown in Figure3NH3PbI3−xClx films were more uniform with higher quality than solution‐processed films. TiO2 was used for the electron transport layer with Spiro‐OMeTAD for the hole transport layer, FTO for the anode, and Ag for the cathode, as used in the well‐known 15% efficiency cell.Perovskites have more suitable bandgaps, larger absorption coefficients, and longer carrier diffusion lengths−2, an open‐circuit voltage of 1.09 V, a fill factor of 0.65, yielding power conversion efficiency of 15.3% as summarized in TableIt is of interest to compare the power conversion efficiency of the designed solar cell to its black counterpart by using our developed device model . First, we benchmark the device model against the well‐known 15% efficiency black perovskite solar cell with a 330 nm thick perovskite film.−2, 76% of the photocurrent of its black counterpart (21.5 mA cm−2), despite the use of a perovskite film that is one order of magnitude thinner. This high photocurrent arises from the enhanced exclusive absorption over the resonant wavelength range which can partially compensate for the lower absorption in the rest of the visible spectrum compared to the black cell. As shown in Figure The designed ultrathin magenta cell achieves a remarkable short‐circuit current of 16.4 mA cmrpart 21. mA cm−2,Figure To demonstrate high‐purity and angle insensitivity features, we compared our designed solar cells to the state‐of‐the‐art angle robust color filters with planar structures.3In summary, a general strategy was both theoretically and experimentally presented to create a new type of macroscopic planar metasurface absorber consisting of a 1D ultrathin planar semiconductor film , a transparent material spacer, and a metallic back reflector. Rather than following the well‐known nano‐optical‐cavity framework, we derived a new formulism to tune not only the optical cavity behaviors but also the dissipative properties to achieve near‐perfect absorption exclusively in the semiconductor film. To demonstrate the power and simplicity of this strategy, a protoype of planar metasurface solar cell was experimentally demonstrated. We further designed high‐efficiency colored perovskite solar cells with planar metasurface structures. The designed solar cells employ ultrathin perovskite films but display reflected colors. Our device model predicts that they can maintain 75% of the efficiency of their black counterparts despite the use of perovskite films that are one order of magnitude thinner. They can also display high‐purity (comparable to those of state‐of‐the‐art color filters) and angle‐insensitive (for viewing angles up to 60°) colors. This work paves the way to miniaturized, planar, and multifunctional solar cells and optoelectronic devices.4Experiment: Electron beam evaporation was used to deposit the multilayer structure shown in Figure 2O3, and Ag were deposited for rates of 0.1, 0.1, and 0.3 nm s−1 and pressures of ≈10−6 torr. Their complex refractive indices were measured using the ellipsometry method. The multilayer structure sample was characterized using scanning electron microscopy and X‐ray photoelectron spectroscopy as shown in Figure 2O5 and Ag were deposited for rates of 0.02 and 0.1 nm s−1 and pressures of ≈10−6 torr by thermal evaporation. ICBA was spin cast. The a‐Si was deposited using the plasma‐enhanced chemical vapor deposition method. The device was tested for AM1.5 simulated sunlight illumination condition. The instrument of Keithley 2400 was used for the data acquisition of the current and voltage.Theory: The expression of the absorption rate per unit length normalized to the incident energy was derived based on the transfer matrix theory. This theory was also used to calculate the reflectivity and the exclusive absorption in the semiconductor film. The device model features coupled electrical and optical modeling. The transport of photogenerated charge carriers is governed by the diffusion and the electrically induced drift equations. The electrostatic potential and carrier concentrations throughout the device were calculated by solving the Poisson and the continuity equations. The photogeneration rate was calculated using the transfer matrix method. The generated charge carriers can recombine via radiative, Auger, Shockley–Read–Hall, and surface mechanisms. The optical parameters from refs. 36,37 and the electrical parameters from refs. 38,39 were used for modeling. The surface recombination velocities for ultrathin colored cells were assumed to be one order of magnitude larger than that for the regular black cell, because the cells with thinner semiconductor active layers have higher interfacial defect level.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


We compared the effects of sodium–glucose cotransporter 2 (SGLT2) inhibitors on renal function in participants with type 2 diabetes and chronic kidney disease (CKD) classified by degree of albuminuria.2) was conducted. Based on the urinary albumin-to-creatinine ratio (UACR) or urinary protein-to-creatinine ratio (UPCR) at the start of SGLT2 inhibitor administration, participants were categorized into three groups: normoalbuminuria , microalbuminuria , and macroalbuminuria . The study outcome was a comparison of the rates of change in renal function evaluated by eGFR at 2 years after starting SGLT2 inhibitor among the three groups.A retrospective review of the clinical records of Japanese participants with type 2 diabetes  < 60 mL/min/1.73 mn = 46), A2 (n = 25), and A3 (n = 16). eGFR was similarly decreased at 2 years before starting SGLT2 inhibitor in all three groups. However, the decline in eGFR was ameliorated at 2 years after starting SGLT2 inhibitor, and eGFR was rather increased in the A1 and A2 groups. Interestingly, the rate of change in eGFR at 2 years after starting SGLT2 inhibitor in the A1 group was significantly higher than that in the A3 group.A total of 87 participants  were categorized into three groups: A1 (These results demonstrate that more favorable effects of SGLT2 inhibitors on renal function were observed in participants with type 2 diabetes and CKD with normoalbuminuria compared with those with macroalbuminuria.Trial registration UMIN-CTR: UMIN000035263. Registered 15 December 2018 Large randomized clinical trials have indicated that sodium–glucose cotransporter 2 (SGLT2) inhibitors can significantly ameliorate renal outcomes in participants with type 2 diabetes at high risk for cardiovascular disease –6. Furth2) in the outpatient center at Kurihara Clinic. Participants were excluded from the study if they had no clear data on eGFR or albuminuria. Participants with type 1 diabetes, and those with poor adherence or interruption of the medication were also excluded. An opt-out consent procedure was used. The study was conducted with approval from the Institutional Review Board of the Japan Clinicians Diabetes Association, and registered with the University Hospital Medical Information Network .The analyzed data were obtained from our previous retrospective observational study . We condBased on the urinary albumin-to-creatinine ratio (UACR) or urinary protein-to-creatinine ratio (UPCR) at the start of SGLT2 inhibitor administration, participants were categorized into three groups: normoalbuminuria , microalbuminuria , and macroalbuminuria  , 11. TheP < 0.05 were considered to indicate statistical significance. All statistical analyses were performed using GraphPad Prism 7  and Microsoft Excel Statistics 2012 for Windows .Data were expressed as mean ± standard deviation or median. Differences in baseline characteristics and changes among the three groups were compared by one-way analysis of variance followed by post hoc Bonferroni test or the Chi square test, as appropriate. Simple linear regression analyses were performed to test for associations between  %∆eGFR + 2y and baseline HbA1c or change in HbA1c. Values of n = 46), A2 (n = 25), and A3 (n = 16). The baseline characteristics of the participants are shown in Table A total of 87 participants  were divided into the three groups as follows Fig. : A1 (n =P = 0.135). However, the decline in eGFR was ameliorated at 2 years after starting SGLT2 inhibitor, and eGFR was rather increased in the A1 and A2 groups. Interestingly,  %∆eGFR + 2y in the A1 group was significantly higher than that in the A3 group  or change in HbA1c .As shown in Fig. The present results indicated that  %∆eGFR + 2y in the A1 group was significantly higher than that in the A3 group. Because baseline HbA1c in the A1 group was lower than that in the A3 group, we assumed that glucose tolerance could affect  %∆eGFR + 2y. However, there was no association between  %∆eGFR + 2y and baseline HbA1c or change in HbA1c at 2 years. These data suggest that glucose tolerance is not related to  %∆eGFR + 2y. Recently, it was reported that the incidence of low eGFR with normoalbuminuria has been increasing in type 2 diabetes , 12, 13.The main limitations of the present study are its retrospective design and relatively small sample. Other limitations are the relatively short follow-up duration and the study endpoint of changes in renal function evaluated by eGFR, rather than hard endpoints such as initiation of renal-replacement therapy or death from renal disease. Further studies are needed to verify our results in a prospective larger cohort for a longer observational period.In conclusion, our study demonstrated that more favorable effects of SGLT2 inhibitors on renal function were observed in participants with type 2 diabetes and CKD with normoalbuminuria than in participants with macroalbuminuria. These findings suggest that the renoprotective effects of SGLT2 inhibitors in participants with CKD can vary depending on the degree of albuminuria.


Fluorescent proteins often result in phototoxicity and cytotoxicity, in particular because some red fluorescent proteins produce and release reactive oxygen species (ROS). The photogeneration of ROS is considered as a detrimental side effect in cellular imaging or is proactively utilized for ablating cancerous tissue. As ancient textiles or biomaterials, silk produced by silkworms can directly be used as fabrics or be processed into materials and structures to host other functional nanomaterials. It is reported that transgenic fusion of far‐red fluorescent protein (mKate2) with silk provides a photosensitizer hybridization platform for photoinducible control of ROS. Taking advantage of green (visible) light activation, native and regenerated mKate2 silk can produce and release superoxide and singlet oxygen, in a comparable manner of visible light‐driven plasmonic photocatalysis. Thus, the genetic expression of mKate2 in silk offers immediately exploitable and scalable photocatalyst‐like biomaterials. It is further envisioned that mKate2 silk can potentially rule out hazardous concerns associated with foreign semiconductor photocatalytic nanomaterials. Visible light‐driven plasmonic photocatalysis, which relies on the combination of semiconductor photocatalysts with metal nanostructures/nanoparticles, has received considerable attention for solar energy conversion and utilization.The phototoxicity of fluorescent proteins, in particular red fluorescent proteins (RFP) is unanimously acknowledged in several different scientific communities; Some of RFPs generate and release ROS upon light excitation, while the exact types of ROS vary among different RFP variants.1O2) via Type II photosensitization reaction, in which energy transfer occurs from the excited triplet state of the fluorescent protein to molecular oxygen.2•−).] Another interesting aspect of ROS resulting from Type I and Type II photosensitization reactions is that the maximum migration (or damage) distance of O2•− and 1O2 is less than 200–300 nm, depending on the surrounding environments.] This relatively short damage distance can be advantageous as a safeguard, given that O2•− and 1O2 are instantaneously reactive and toxic. Importantly, the resultant ROS  generated by plasmonic photocatalysis using visible light is the same as that of RFP photosensitization reactions.]Some fluorescent proteins participate in Type I and Type II photosensitization reactions.In this work, we introduce biological hybridization of far‐red fluorescent proteins and natural proteins  for a new class of genetically encoded photosensitization that can be activated by visible (or solar) light, producing selective ROS in a similar manner of plasmonic photocatalysis. Direct detection of ROS is known to be highly challenging, because ROS is extremely reactive and unstable. Thus, we implement several different approaches using turn‐on/‐off fluorescent radical probes and physical quenchers/scavengers to experimentally validate ROS generated by transgenic RFP silk upon green light activation. We demonstrate that transgenic RFP silk can be mass‐produced by scalable and continuous manufacturing using the currently available textile infrastructure. Using the polymeric nature of silk, transgenic RFP silk is further processed into nanomaterials and nanostructures in a variety of forms. The use of plasmonic photocatalyst‐like proteins can overcome the limitation of potential adverse effects associated with foreign synthesized nanoparticles. We also envision that this bioreactor approach could potentially offer an alternative green manufacturing strategy for next generation photocatalysts.Bombyx mori)  Figure1a. Silac. ThiWe choose mKate2, which is a far‐red monomeric fluorescent protein.[[qv: 5a]] From a phototoxicity standpoint, mKate and mKate2 are widely considered as one of the cytotoxic standards.[[qv: 5b–d]] From a protein structural standpoint, the phototoxic action of mKate is commonly acknowledged to originate from a cleft‐like opening channel filled with water molecules inside, allowing for enhanced generation and release of ROS. Specifically, mKate has a cleft‐like β‐barrel frame between β sheets β7 and β10), resulting in relatively high phototoxicity.] Several other fluorescent proteins, including KillerRed,0, resultFigureex = 532 nm and optical intensity ≈0.2 mW mm−2; Supporting Information) at the ambient room temperature. Although this crude method is not specific to particular types of ROS, the photodegradation of methylene blue serves as a standard for validating photocatalysis. However, silk has a strong affinity to organic molecules and metal ions.] Thus, the loss of blue color in a methylene blue solution containing mKate2 silk is attributable to the infiltration  of methylene blue to silk fibers as well as the photolysis of methylene blue itself by green light. In this respect, we performed separate degradation measurements to account for the adsorption of methylene blue to silk under a dark condition  and the photolysis of methylene blue without any silk disks . After factoring out these confounding effects, the contribution of ROS generated by the mKate2 silk is significant; a linear fit between ln(tC/C0) of methylene blue by mKate2 silk and the irradiation time t results in an apparent pseudo‐first‐order rate constant (kapp) value of 2.46 × 10−4 min−1  of 30 nm and optical intensity ≈ 0.02 mW mm−2; Supporting Information), which is ≈10 times weaker than that of the green laser source above, is irradiated on the surface of white silk and mKate2 silk for 30 and 60 min at the ambient room temperature. Such green light activation is not only accessible from sunlight, but also belongs to the peak solar radiation spectrum. Dark controls are also maintained without any light irradiation. Colony‐forming unit (CFU) counts show a statistically significant difference only in bacterial inactivation between irradiated (Light ON) and un‐irradiated (Light OFF) mKate2 silk for 60 min  . The survival rate of E. coli from mKate2 silk under weak green light activation (Light ON) is reduced to 45%, compared with the corresponding dark controls (Light OFF). This result supports the idea of green light‐activated genetically encoded photosensitization as an alternative ROS generation route, completely avoiding the use of photocatalytic semiconductor nanoparticles.As a model system of testing ROS production, we also examine the phototoxicity of mKate2 silk on λex = 532 nm and optical intensity ≈ 0.2 mW mm−2) amino]‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (TEMPO‐9‐ac) is commonly used to sense O2•−.ex ≈ 360 nm and λem ≈ 440 nm) are detected in two different configurations : (i) The turn‐on fluorescent radical probes on the mKate2 silk surface are diffused in the TEMPO‐9‐ac solution. In Figure 2•− released from KillerRed, which is one of the highly phototoxic RFP variants.1O2 generated by mKate2 silk via Type II photosensitization reaction under the same green light activation, using 9,10‐anthracenediyl‐bis(methylene)dimalonic acid (ABDA) as a radical probe. While the original state of ABDA emits fluorescence (λex ≈ 380 nm and λem ≈ 431 nm), ABDA reacts with 1O2 to yield endoperoxide as a turn‐off fluorescent radical probe, reducing its fluorescent intensity.1O2.We further investigate specific types of ROS produced by mKate2 silk upon green light activation .2•− and 1O2 production. In other words, the uptake of local surrounding ROS (O2•− and 1O2) prevents mKate2 silk from being photodamaged, which is manifested by the relatively sustained fluorescent intensity of mKate2 silk. In a mixed solution of TEMPO‐9‐ac and ABDA, the fluorescent emission of mKate2 silk is further maintained  and 1O2  .Using fluorogenic scavengers, we additionally validate the generation of Od Figure e. We alsFigure2•− and 1O2 from the regenerated mKate2 silk products is also detected using TEMPO‐9‐ac and ABDA, respectively , the fluorescent signal of TEMPO‐9‐ac increases, while that of ABDA decreases, supporting the two types of ROS generation. Similarly, the photodegradation of methylene blue by the mKate2 silk film results in kapp = 1.12 × 10−3 min−1 under green light irradiation, after factoring out the confounding effects  .The direct use of silk fibers produced by silkworms has its own advantage as utilized in the textile industry, because the transgenic silk has the comparable mechanical properties to wild‐type silk to weave fabrics . Silk fibroin can further be processed into polymeric materials for fabricating artificially engineered biomaterials and optical materials in a variety of forms with biocompatibility and bioabsorbability.[[qv: 13b–f]] However, the conventional fibroin processing methods are inappropriate for mKate2 silk,[[qv: 13c–e]] because fluorescent proteins are highly susceptible to denaturation from high temperature and pH values.] In our case, to minimize heat‐induced denaturation of mKate2, mKate2 silk fibroin is extracted from silk cocoons at low temperature of 45 °C, assisted by alcalase enzyme and dithiothreitol (DTT) treatments.[[qv: 13f]] A reductase, such as DTT, is beneficial for renaturing the protein structure by reducing the disulfide bonds of proteins and peptides in a solvent.From an ecological standpoint, our results may suggest that the primary purpose of fluorescent proteins in nature could be photoinducible ROS generation, while the fluorescent emission may be a secondary consequence. From a mechanistic standpoint, ROS generation from fluorescent proteins is known to involve long‐range electron transfer via two possible mechanisms of direct tunneling and hopping inside fluorescent proteins.] The current understanding of this mechanism is based on quantum mechanics, because electron tunneling over such a long distance of 1.5–3 nm is typically impossible in vacuum.In conclusion, the reported hybridization of mKate2 and silk using genetically engineered silkworms can offer several pivotal advantages. Without a need of additional nanoconjugations , RFP can be excited by solar (visible) and green light, avoiding the most common carcinogen exposure of ultraviolet light. Both fluorescent proteins and silk are degradable and digestible,Removal of Sericin in Silk : For effective generation and release of ROS from mKate2 silk, it was critical to remove the outermost layer  of silk fibers. Sericin was removed using a degumming process. The outer sericin layer is commonly removed to improve the color, sheen, and texture of silk in the silk textile industry. However, conventional sericin removal methods are inappropriate for mKate2 silk, because these involve a boiling process in an aqueous solution.[[qv: 13c–e]] In this case, mKate2 silk cocoons were soaked in a prewarmed mixture solution of sodium carbonate  and Triton X100 (0.1%) at low temperature of <60 °C under a vacuum pressure. During the degumming process, low pressure treatments (620 mmHg) were repeated several times to uniformly infiltrate the solution between silk fibers to remove most sericin. The degummed mKate2 silk cocoons were dried in dark under the ambient air conditions.Photodegradation of Methylene Blue as General Photocatalytic Quantification: Photodegradation of methylene blue, resulting from ROS generated by mKate2 silk under green light activation, was quantified. For mKate2 silk specimens, silk cocoons were punched into 5 mm diameter disks with a thickness of ≈400 µm. Methylene blue solutions (1 mL 0.05 wt% methylene blue in 14 mL deionized water) containing 12 silk disks  were prepared. To reach the adsorption–desorption equilibrium in each test, the silk disks were stirred with 400 rpm in dark for 2 h. Then, the silk disks were irradiated by green light  for 4 h, while being stirred. Aliquots (0.5 mL) were collected repeatedly with a fixed time interval and the spectral absorption of methylene blue was measured using a fiber bundle‐coupled spectrometer with a white‐light tungsten halogen source. To exactly quantify the photocatalytic activity of mKate2 silk only, separate degradation tests of methylene blue were also carried out to factor out two confounding effects: (i) the adsorption of methylene blue to silk under a dark condition  and (ii) the photolysis of methylene blue without any silk disks by green light. For each elapsed irradiation time, a relative concentration tC/C0 of methylene blue was calculated using the absorption spectrum peak values tC at λ = 668 nm normalized by the absorption value C0 before light irradiation  = −kappt, where kapp is the rate constant (min−1) and t is the irradiation time  and two light conditions  were conducted. These experiments were repeated for two different irradiation times of 30 and 60 min. Each bacterial inactivation experiment was performed in three assays with four replicates (n = 12) in each group for statistical analyses. DH5α E. coli cells were grown in a Luria–Bertani (LB) medium at 37 °C in a shaking incubator to an optical density at 600 nm (OD600) of 2.5 (≈2 × 109 cells mL−1). The culture was diluted tenfold and subsequently white silk and mKate2 silk disks (diameter = 6 mm) were placed on the culture. After incubation at 37 °C for 60 min, each silk disk was dried in dark for 30 min. For optical excitation of mKate2, the silk disks on a hydrated filter paper were irradiated with the green LED source  for 30 and 60 min at the ambient room temperature, including white silk disks for comparisons. Without any irradiation, both white silk and mKate2 silk disks were kept in dark under the same conditions as two different control groups. After green light activation, each silk disk was transferred to a phosphate buffered saline  solution (1 mL) and E. coli cells were eluted by shaking incubation for 60 min. To achieve a reasonable number of surviving cells for counting the colonies, the eluted cells were diluted up to 1000‐fold, were plated on the LB agar, and were incubated overnight at 37 °C. CFU from the mKate2 silk disk irradiated under weak green light for 60 min was clearly lower than that of the mKate2 silk disk in dark  and Singlet Oxygen (1O2) Using Fluorescent Radical ProbesDetection of Superoxide .ex ≈ 380 nm and λem ≈ 431 nm).1O2, it is converted to an endoperoxide form that leads to a decrease in the fluorescent intensity. In this study, TEMPO‐9‐ac and ABDA were initially dissolved in dimethyl sulfoxide and were diluted in PBS, respectively, resulting in each solution containing TEMPO‐9‐ac (20 × 10–6m) or ABDA (20 × 10–6m). In each measurement, 12 silk disks  or regenerated silk films were immersed in a TEMPO‐9‐ac or ABDA solution with stirring of 400 rpm. Because water‐soluble molecules were easily smeared inside silk fibers, the adsorption–desorption equilibrium was achieved prior to green light activation; the silk disks were kept in the solution with stirring of 400 rpm in dark for 2 h at least. Turn‐on fluorescent signals of TEMPO‐9‐ac solutions and turn‐off fluorescent signals of ABDA solutions were spectrofluorimetrically monitored using a spectrometer. Turn‐on fluorescence  from the mKate2 silk disks was also imaged and was measured using a custom‐build mesoscopic (between microscopic and macroscopic) imaging setup .]2•−) and Singlet Oxygen (1O2) Using ScavengersDetection of Superoxide .ex = 532 nm and optical intensity ≈ 0.2 mW mm−2), the photobleaching effect of mKate2 silk was reduced in the presence of TEMPO‐9‐ac (20 × 10–6m); the fluorescent emission was relatively maintained over the irradiation time in the presence of the physical scavenger of O2•−. Similarly, 1O2 generation was detected by the maintained fluorescent intensity of mKate2 silk in the presence of ABDA (20 × 10–6m). In addition, reduced photobleaching rates of mKate2 silk were confirmed using NBT (200 × 10–6m) and NaN3 (200 × 10−3m), which are often used as a scavenger of O2•− and 1O2, respectively .Regeneration of mKate2 Silk: To use the polymeric nature of silk, mKate2 silk was regenerated by extracting mKate2 silk fibroin from silk cocoons. mKate2 silk cocoons were cut to pieces with sizes less than 5 mm and were heated for 4 h at ≈45 °C in a aqueous solution of NaHCO3 (50 × 10−3m) with alcalase (1.5 mL L−1) with stirring of 400 rpm. Subsequently, the silk fibers were washed with deionized water (≈35 °C) several times and were dried in dark under the ambient conditions for 24 h. It was also noted that conventional fibroin dissolution methods are not ideal for mKate2 silk, because these require chemical‐based solution treatments at temperature of 60 °C.[[qv: 13c–e]] In this case, the silk fibers were completely dissolved in a lithium bromide  solution with DTT (1 × 10−3m) at 45 °C. The dissolved solution was filtered through a miracloth and was dialyzed with deionized water for 2 d to remove the remaining salt. The final concentration of mKate2 silk fibroin in the solution was ≈4–5% (w v−1). When the same method was followed for wild‐type white silk under the identical conditions, a similar final concentration of silk fibroin was obtained. The solution was stored at 4 °C in dark before use. The fabrication process of the mKate2 silk solution was carried out in dark environment to minimize photobleaching of mKate2 in silk by the room light. To form silk films, the solution was dried at 30 °C for 12 h in an oven.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


A calix[4]pyrrole tetraurea, a polar guest and methyltrioctylammonium chloride quantitatively self-assemble in two different complexes in response to the components' stoichiometry. 1 with a polar neutral guest, trimethylamine N-oxide or beta-alanine betaine, and methyltrioctylammonium chloride (MTOACl) salt into two supramolecular architectures which differ in morphology and stoichiometry. Whereas an equimolar solution of tetraurea calixpyrrole 1, polar guest and MTOACl produces a four-particle inclusion assembly, a mixture of the same components in a 2 : 1 : 1 molar ratio induces the formation of a dimeric capsular assembly displaying multiple guests which are orderly co-encapsulated. The influence of other polar guests and ammonium salts on the self-assembly process is also described.In this work, we describe the stoichiometrically controlled self-assembly process of tetraurea calix[4]pyrrole  Self-assembled molecular capsules based on hydrogen bonding interactions are a well-known class of synthetic molecular containers. They enclose small spaces in which molecules are confined, contiguously enclosed and completely isolated from the bulk solvent. However, the extensive use of aromatic groups in the scaffolds of the capsule’s components challenges the inclusion of polar groups that can be presented to the encapsulated guests.In contrast, biological receptors contain a mixture of polar and hydrophobic residues converging in their binding sites. The formation of ordered encapsulation complexes and the encapsulation of polar guests require the inclusion of polar functions in the cavity of the container.3 solution tetraurea aryl-extended calix[4]pyrrole 1 dimerizes via encapsulating one molecule of 4,4′-bipyridine-N,N′-dioxide 2 to yield the capsular assembly 2⊂12 stabilized by a cyclic array of 16 hydrogen bonds.12 featured a polar interior and established multiple directional interactions (hydrogen bonds) with the encapsulated guest. Later on, we demonstrated the pairwise encapsulation of trimethylamine N-oxide 3 and trimethylphosphine oxide 4 in the same dimeric molecular container 12.12 was also responsible for the selective binding of 3 and the formation of ordered encapsulation complexes, (3·3)⊂12 and (3·4)⊂12, respectively.A few years ago, we reported that in CDClsyn–syn form of the urea group have been extensively exploited for the recognition of anions.1 bearing two different binding sites for anions and one binding site for cations undoubtedly qualifies as a multitopic ion-pair receptor.In non-polar solvents, calix[4]pyrroles are known to function as heteroditopic receptors for ion-pairs.1 with a polar neutral guest, trimethylamine N-oxide 3 or beta-alanine betaine 12, and methyltrioctylammonium chloride salt , in two diverse supramolecular architectures which differ in morphology and stoichiometry. On the one hand, we observed that 1 dimerized through co-encapsulating the chloride anion and the polar guest to afford a five particle capsular assembly (Cl–·polar guest)⊂12·MTOA+ when a 2 : 1 : 1 molar ratio of the components  was used. On the other hand, a mixture of the components in a 1 : 1 : 1 molar ratio produced exclusively a four particle inclusion complex, polar guest⊂1·MTOACl.In this work, we describe the stoichiometrically controlled self-assembly process of tetraurea calix[4]pyrrole 1H NMR spectrum of tetraurea 1 in non-polar solvents like CDCl3 or CD2Cl2 shows broad and ill-resolved signals ⊂12. We calculated a similar packing coefficient for the encapsulation of the capsular assembly 2⊂12.N-oxide 5 to CDCl3 or CD2Cl2 suspensions of tetraurea 1 produced the formation of a white precipitate.1H NMR analysis of the liquid phase testified the total absence of detectable proton signals. The solid precipitate was filtered and dissolved in DMSO-d6. The 1H NMR spectrum of the solution indicated the presence of diagnostic signals for 1 and 5, free in solution, in an exact 1 : 1 ratio.‡The  We hypothesized that pyridine N-oxide 5 was indeed included in the deep aromatic cavity of tetraurea 1 but the resulting 5⊂1 complex did not dimerize to form the expected (5·5)⊂12 capsular assembly. Instead, the initially formed 5⊂1 complex experienced a strong aggregation process, probably mediated by hydrogen bonding interactions between their urea groups, yielding polymeric aggregates that precipitated out of solution. Inspired by the concept of tuning the sol–gel properties of urea derivatives through anion binding,The s Fig. S1. This is6 or TBACl, 7) to the liquid samples (CD2Cl2 or CDCl3 solvent) containing the white precipitate, produced a transparent solution after shaking the mixture for several minutes. The 1H NMR spectrum of the solution showed sharp proton signals indicative of the formation of a well-defined assembly with C4v symmetry (c) of 1 were downfield shifted. In contrast, the protons ortho and meta (H1 and H2) with respect to the nitrogen atom of the bound pyridine N-oxide 5 moved upfield with respect to the corresponding signals in free 5. Taken together, these observations indicated the inclusion of 5 in the deep aromatic cavity of the cone conformation of 1. The inclusion process was driven by the formation of four hydrogen bonds between the oxygen atom of the N-oxide 5 and the pyrrole NHs of 1. However, the location and binding geometry of the ammonium chloride salt (ion-pair) in the formed aggregate remained to be clarified. In the specific case of MTOACl 6, we noticed that the N-methyl group of the organic cation appeared at δ = 0.74 ppm (Δδ = –2.6 ppm). This intense upfield shift supported its inclusion in the shallow π-cavity offered by the cone conformation of 1, opposite to the included N-oxide 5  in the formed aggregate appeared downfield shifted compared to the signals in the free mono-urea reference compound 9  and MTOABr (8) produced identical results  for the three counterparts that is significantly smaller than for the free counterparts  experiments. The calculated binding constant values are summarized in Using 9 and 10 with N-oxide 11, and MTOACl 6 (5⊂1·MTOA+·Cl– complex: (a) preferential inclusion of the pyridine N-oxide 5 in the deep aromatic cavity of the calix[4]pyrrole 1 and (b) higher affinity of the chloride ion-pairs (MTOACl) for the urea groups. The high selectivity in binding MTOACl, in comparison to TBACl, displayed by calix[4]pyrrole 10 is derived from the known heteroditopic nature of this type of receptors9 being a monotopic anion receptor exclusively recognized the chloride and did not show any sign of selectivity in the binding of MTOACl 6vs. TBACl 7.The relative order of interaction strengths measured for the 1 : 1 complexes of the model receptors MTOACl 6  is in co3 suspension of tetraurea 1 produced a transparent solution, but contrary to our expectations it did not induce the formation of a capsular aggregate i.e. (Cl–·Cl–)⊂12·(MTOA+)2. The observation of broad signals in the 1H NMR spectrum of the mixture hinted at the formation of stoichiometrically and/or structurally ill-defined aggregates. This negative result prompted us to study the dimerization of 1 induced through co-encapsulation of a suitable N-oxide and the chloride anion. Recently, we reported the quantitative pairwise encapsulation of trimethylamine N-oxide 3 yielding a capsular assembly (3·3)⊂12.N-oxide 3 as the co-encapsulation guest with chloride in the polar cavity of the 12 capsule. We expected that a capsular assembly of the type (N-oxide·Cl–)⊂12·MTOA+ would allow the fine tuning of the cavity contents and also eliminate the plausible electrostatic repulsion between two negatively charged encapsulated guests. We considered that the preferential assembly of the (N-oxide·Cl–)⊂12·MTOA+ encapsulation complex would require working under strict stoichiometric control of the components.The addition of 1 equiv. of MTOACl to a CDCl1H NMR spectrum ⊂12  were also visible for the NHs of the urea alpha to the meso-phenyl groups of 1 (Hg). The chemical shift values of these NHs suggested their involvement in hydrogen bonding interactions. The methyl groups of 3 were upfield shifted at δ = 0.90 ppm and suggested that the position of the N-oxide was deep in the aromatic cavity of 1. Taken together, these observations hinted at the formation of a capsular dimeric assembly (3·Cl–)⊂12·MTOA+.The spectrum  of an eq Fig. S28. We obse12 and rendered the two hemispheres chemically non-equivalent. Another independent element of asymmetry present in the (3·Cl–)⊂12·MTOA+ capsular assembly was derived from the unidirectional orientation of the urea groups that was kinetically stable on the 1H NMR timescale. This resulted in the existence of the five particle capsular aggregate (3·Cl–)⊂12·MTOA+ as a pair of enantiomers. The latter asymmetry was expressed by the observation of resolved diastereotopic signals for some of the aromatic protons belonging to the same hemisphere in 12. Other diastereotopic protons appeared as broad signals. Because the two hemispheres in (3·Cl–)⊂12·MTOA+ are chemically non-equivalent, a total of eight diastereotopic signals can be expected for the meso-phenyl protons (Ha and Hb) and four for the benzylic protons (Hi). Nevertheless, proton signal overlapping and broadening hampered the observation of separate signals for the eight aromatic protons. The number of proton signals observed for the capsule was in agreement with a C4 symmetry, in contrast to the C4v symmetry determined for the inclusion complex 5⊂1·MTOA+·Cl–.The co-encapsulation of two different guests produced a desymmetrisation of the capsular assembly N-methyl group of the co-bound MTOA cation resonated as a broad singlet at δ = 0.38 ppm testifying its inclusion in the exo-cavity defined by the four pyrrole rings opposite to the bound chloride. A ROESY experiment provided additional evidence for the capsule formation ⊂12·MTOA+ capsule assembly provided a value of 39%. Most likely, a molecule of the solvent was co-encapsulated with the two guests to achieve a packing coefficient closer to the optimum value of 55%. Indeed, the recalculation of the packing coefficient value considering the co-encapsulation of the three guests, 3, Cl– and CDCl3, returned a value of 58% (1H NMR spectra of the capsular aggregates (3·Cl–)⊂12·MTOA+ recorded in CDCl3 or CD2Cl2 solutions that cannot be explained by a simple change in solvent  compared to that experienced (Δδ = –3.90 ppm) in its encapsulation by a tetraurea calix[4]pyrrole/tetraurea calix[4]arene bis[2]catenaned capsule.12 producing a single constellational isomerN-oxide 3 and chloride, occupied the polar ends of the container with the CHCl3 molecule sandwiched between them. Previous examples of constellational encapsulation isomers of dimeric capsules have always been produced as mixtures of isomers.36The calculation of the packing coefficient for the ⊂12·MTOA+ capsular assembly is not formed in solution due to a low packing coefficient. In silico, the co-encapsulation of a solvent molecule with the two guests 5 and the chloride disrupted the capsular structure.Disappointingly, the  Fig. S10.¶¶The uN-oxide 3, stimulated us to assess the self-assembly properties of the system using a 1 : 1 : 1 molar ratio of trimethylamine N-oxide 3, tetraurea 1, and MTOACl. The analysis of the equimolar mixture using 1H NMR spectroscopy revealed the presence of a main set of proton signals for the bound tetraurea 1 that were almost coincident with those observed for the 5⊂1·MTOA+·Cl– complex ⊂12·MTOA+ were still visible. In short, the system self-sorted itself in the almost exclusive formation of the inclusion complex 3⊂1·MTOA+·Cl– in response to the equimolar mixture of components. Notably, the methyl protons for bound 3 in the 3⊂1·MTOA+·Cl– complex were upfield shifted at δ = 0.75 ppm. This chemical shift value was markedly different from that observed for the same methyl protons of bound 3 in the capsular assemblies (3·Cl–)⊂12·MTOA+ (δ = 0.90 ppm) and (3·3)⊂12 (δ = 0.54 ppm) and provided support for the presence of different magnetic environments. The diffusion coefficient values calculated for the two species, the capsular assembly (3·Cl–)⊂12·MTOA+ and the inclusion complex 3⊂1·MTOA+·Cl–, through performing DOSY experiments on CDCl3 solutions of tetraurea 1, trimethylamine N-oxide and MTOACl with molar ratios of 2 : 1 : 1 and 1 : 1 : 1, respectively, were in complete agreement with their difference in size  for obtaining the (3·Cl–)⊂12·TBA+ capsular aggregate, we observed the presence of the encapsulation complex (3·3)⊂12 in combination with other unassigned aggregates ⊂12·A+.The change of MTOACl by TBACl eliminated the observed stoichiometric control of the self-assembly process. On the one hand, the equimolar mixture of trimethylamine  Fig. S37. In summ1 in a host separated ion-pair geometry, Cl–⊂1·MTOA+, with the chloride included in the aromatic cavity of the calix[4]pyrrole and the N-methyl group of the co-bound cation located in the shallow aromatic cavity opposite to the bound anion. In this binding geometry, the urea arms of 1 in Cl–⊂1·MTOA+ are available to engage in hydrogen bonding interactions with their counterparts in the N-oxide inclusion complex, 3⊂1. The net result being the assembly of the encapsulation complex (3·Cl–)⊂12·MTOA+ as the almost exclusive species in solution.Probably, the MTOACl is preferentially bound by tetraurea 1 in a close-contact binding geometry, 1·TBACl, through establishing hydrogen bonding interactions between the chloride and the urea groups (1 exists between the chloride in TBACl and the urea groups of the 3⊂1 complex. For this reason and to a certain extent the dimerization of the 3⊂1 complex yielding the encapsulation complex (3·3)⊂12 competes with the oligomerization of 1·TBACl. The result of the equilibria produced no detectable signals for the encapsulation complex (3·Cl–)⊂12·TBA+.Conversely, TBACl is bound better by the tetraurea a groups . That is12. Specifically, the beta-alanine betaine 12 was a nice fit with respect to size, shape and chemical surface to the inner space of the capsule that was left after chloride encapsulation. Calix[4]pyrroles are known to be good receptors for carboxylates.12 can establish hydrogen bonds with the endo-directed pyrrole NHs and the trimethylammonium group forms favorable coulombic interactions with the co-encapsulated chloride anion. We calculated a packing coefficient value for the encapsulation complex (12·Cl–)⊂12·MTOA+ of 55%.We reasoned that certain betaines could also be appropriate co-encapsulation guests with chloride in the dimeric capsule 3 solution of tetraurea 1, betaine 12 and MTOACl in a 2 : 1 : 1 molar ratio produced a 1H NMR spectrum with diagnostic signals of the formation of the encapsulation complex (12·Cl–)⊂12·MTOA+ (N-oxide 3 in (3·Cl–)⊂12·MTOA+, one of the NHs moved upfield and the other downfield ⊂12·MTOA+ capsule. A 2D ROESY experiment allowed the assignment of the signals for the two methylene protons of the encapsulated 12. The bound trimethylalkylammonium group moved upfield and showed intermolecular nOes with protons in the meso-phenyl protons  and the urea groups of the calix[4]pyrrole units. Altogether, these observations supported the co-encapsulation of 12 and chloride in 12. The N-methyl group of the MTOA co-bound cation resonated at δ = 0.34 ppm owing to its location in the base of the calix[4]pyrrole unit opposite to the encapsulated chloride. A DOSY NMR experiment performed on a CDCl3 solution of tetraurea 1, betaine 12 and MTOACl in a 2 : 1 : 1 molar ratio provides a diffusion coefficient value in total agreement with that measured for the capsular assembly (3·Cl–)⊂12·MTOA+ ⊂12·MTOA, in which the betaine and bromide are co-encapsulated. However, the higher packing coefficient (58%) for the complex reduces its stability and also the proton signals of the four-particle 1 : 1 : 1 complex 12⊂1·MTOA+·Br– can be observed as a minor species (ESI).‖Gratifyingly, the combination of tetraurea A CDCl12·MTOA+ . In compownfield . This ob Fig. S59.‖‖Grati12·Cl–)⊂12·TBA+ to a minimum extent ⊂12·MTOA+ and this is in agreement with the formation of the inclusion complex 12⊂1·MTOA+·Cl–. The methylene protons alpha to the carboxylate and alpha to the ammonium group resonate less upfield than in the capsular assembly ⊂12·MTOA+ was 53%, indicating a reduced size complementarity for the co-encapsulation of 13, instead of 12, with chloride. In agreement with this calculation, the 1H NMR spectrum of a solution containing a mixture of tetraurea 1, betaine 13 and MTOACl in a 2 : 1 : 1 molar ratio was composed of signals for the capsular assembly (13·Cl–)⊂12·MTOA+ and the inclusion complex 13⊂1·MTOA+·Cl–. By integrating selected proton signals in each one of the two aggregates we determined that they were present in solution in a ratio close to 1 : 1.13·Cl–)⊂12·MTOA+ and the 1 : 1 : 1 complex 13⊂1·MTOA+·Cl– evidenced the difference in size of the two aggregates (13 resonated at 0.70 ppm in the capsular assembly (13·Cl–)⊂12·MTOA+ whereas those in the 1 : 1 : 1 inclusion complex 13⊂1·MTOA+·Cl– appeared less upfield shifted at δ = 0.84 ppm. The N-methyl protons of the MTOA cation resonated as a single signal at δ = 0.34 ppm. The resulting upfield shift indicated their placement at the shallow cavity of the calixpyrrole. The diffusion coefficient value calculated for the N-methyl cation represented the weighted average of the two species indicating that the cation was involved in an exchange process that was fast on the DOSY timescale. On the contrary, the chemical exchange between the calix[4]pyrrole units involved in the two species was slow on the EXSY timescale. The use of an equimolar mixture of the components produced the 1 : 1 : 1 complex as the exclusive species in solution  the use of a methyltrialkylammonium cation as the chloride counter-ion and (b) that the sum of volumes of the encapsulated guests was adequate to fill a little more than half of the capsule's interior. We have shown rare examples of the ordered encapsulation assemblies of multiple guests. The introduction of polar groups in the capsule interior and the establishment of directional interactions provided unprecedented ordered encapsulation complexes of multiple polar guests displaying high kinetic and thermodynamic stability.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Sci., 2014, 5, 2456–2460.Correction for ‘Pattern-based sensing of aminoglycosides with fluorescent amphiphiles’ by Ziya Köstereli  This compound should feature a C17 instead of a C18 alkane side chain.The publication and the ESIMain text:1) 1st page, right side, line 3 from the bottom, “octadecane” should be replaced by “heptadecane”.2 depicted in Scheme 3 should feature a heptadecane and not an octadecane side chain.2) Compound ESIB and 2 depicted in the reaction scheme should both feature a heptadecane and not an octadecane side chain.1) The compounds The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.Supplementary informationClick here for additional data file.


Red Lettuce Leaves 1 to RLL14:  to RLL4) that contribute to colour variations in lettuce. The RLL1 gene encodes a bHLH transcription factor, and a 5‐bp deletion in some cultivars abolishes its function to activate the anthocyanin biosynthesis pathway. The RLL2 gene encodes an R2R3‐MYB transcription factor, which was derived from a duplication followed by mutations in its promoter region. The RLL3 gene encodes an R2‐MYB transcription factor, which down‐regulates anthocyanin biosynthesis through competing with RLL2 for interaction with RLL1; a mis‐sense mutation compromises the capacity of RLL3 to bind RLL1. The RLL4 gene encodes a WD‐40 transcription factor, homologous to the RUP genes suppressing the UV‐B signal transduction pathway in Arabidopsis; a mis‐sense mutation in rll4 attenuates its suppressing function, leading to a high concentration of anthocyanins. Sequence analysis of the RLL1‐RLL4 genes from wild and cultivated lettuce showed that their function‐changing mutations occurred after domestication. The mutations in rll1 disrupt anthocyanin biosynthesis, while the mutations in RLL2, rll3 and rll4 activate anthocyanin biosynthesis, showing disruptive selection for leaf colour during domestication of lettuce. The characterization of multiple polymorphic genes in this study provides the necessary molecular resources for the rational breeding of lettuce cultivars with distinct levels of red pigments and green cultivars with high levels of health‐promoting flavonoids.Anthocyanins protect plants from biotic and abiotic stressors and provide great health benefits to consumers. In this study, we cloned four genes ( Anthocyanins may also accumulate in leaves to protect the photosynthetic apparatus and photolabile compounds from ultraviolet radiation Gould, . The accet al., et al., et al., et al., et al., et al., HY5, which regulates the expression of genes in the anthocyanin biosynthesis pathway , which catalyse the production of dihydroflavonols and late biosynthetic genes (LBGs), which lead to the biosynthesis of proanthocyanidins and anthocyanins  and high‐throughput sequencing has been proven to be an efficient and cost‐effective method for the genetic analysis of important traits in crops (Dou Lactuca sativa) is one of the most important vegetable crops worldwide. Lettuce genomics has advanced rapidly in recent years , cyanidin‐3‐O‐ and cyanidin‐3‐O‐β‐glucopyranoside, respectively  was crossed with a cultivar of stem lettuce . The F2 population, 50 plants with the most intense red colour were chosen for a ‘red pool’ and 50 plants with green leaves were randomly chosen for a ‘green pool’. Equal amounts of leaf tissue were collected from each individual and pooled. RNA was extracted from each contrasting pool of leaves. The RNA‐seq libraries were sequenced, and reads were mapped to the lettuce genome sequence v8  and Red Lettuce Leaf 2 (RLL2), were shown to be statistically associated with variations in leaf colour (i.e. green or red leaves) in this population. F2 individuals that are homozygous for the Y37 allele at the RLL1 locus (i.e. rll1/rll1) always have green leaves. Similarly, most F2 individuals that are homozygous for the Y37 allele at the RLL2 locus (i.e. rll2/rll2) have green leaves. The combination of the RLL1 and RLL2 loci explained the colour variation (i.e. green or red leaves) for the majority of individuals in the F2 population. Based on these data, we concluded that RLL1 and RLL2 are two qualitative loci.From this Fon Table . Two locRLL1 gene, F2 individuals with RLL1/rll1 and RLL2/RLL2 genotypes were selfed to generate F2:3 families. One F2:3 family (S9) with red to green phenotypes segregating in an approximately 3:1 ratio was chosen for fine mapping of the RLL1 gene  , the RLL1 gene was mapped between 335.69 and 337.92 Mb on chromosome 5 . Transformation of a green individual from the segregating population with the wild‐type RLL1 gene from the red parent (S1) yielded transgenic progeny with red leaves. These data indicate that this bHLH‐encoding gene is RLL1 and that RLL1 promotes the accumulation of anthocyanins in lettuce  are associated with anthocyanin biosynthesis  in this subpopulation. The differences in their expression were confirmed with a qRT‐PCR analysis that utilized near‐isogenic lines (NILs) that differed in the genomic region containing the RLL1 and rll1 alleles  down‐regulates the expression of multiple genes in the biosynthetic pathway of anthocyanin and leads to constitutively green leaves.The RNA‐seq data from the red and green pools that were prepared from the S9 Fs Figure . Y1H asss Figure . Therefo2:3 family (Q16) that was homozygous for RLL1 (from the red parent) and that was segregating the RLL2 locus was used to clone the RLL2 gene. The Q16 F2:3 family had 4058 red and 1372 green individuals . A BSA + RNA‐seq experiment confirmed that a single locus on chromosome 5 was responsible for the segregating leaf colour in this family  were amplified from the genome of the red parent, which were all mapped to the same locus using gene‐specific markers  was found in the genome of the green parent, which is identical to the gene in the reference genome. Only the RLL2A gene from the red parent is highly expressed . The differential expression of these four genes was confirmed using qRT‐PCR  genotypes have green leaves, some individuals became light red when the seedlings were approximately 1 month old  in the CDS of this MYB gene cause amino acid substitutions (C42Y and W52S)  interacted with RLL2 (R2R3‐MYB) and LsTTG1, the WD40 component in the MBW complex , which provided evidence for an additional locus (RLL4) controlling leaf colour  in the conserved WD40 domain  was also found in Arabidopsis  was not affected by the RLL4 gene . The RLL3 gene is a negative regulator of anthocyanin biosynthesis. The mis‐sense mutation in RLL3 that is responsible for the pigmentation phenotype was not found in the wild genotypes, but was found in three cultivars of loose leaf, eight romaine, three butterhead and four crisphead. Similarly, the causal mis‐sense mutation responsible for the V134A substitution in RLL4 was found only in five cultivated genotypes. Therefore, the loss‐of‐function mutations in the RLL3 and RLL4 genes that promote the accumulation of anthocyanins in lettuce leaves were selected after domestication.We also investigated the evolution of the three genes that promote the accumulation of anthocyanins in lettuce . The other 41 genotypes have two or more homologs of RLL2 that could be grouped into three distinct RLL2 haplotypes. Genotypes CGN05092, CGN09365 and CGN13325 representing the three haplotypes were chosen to characterize their RLL2 homologs. TA cloning and subsequent sequencing discovered three RLL2 homologs from CGN05092, CGN09365 and CGN13325, respectively. Consequently, a total of 34 distinct RLL2 genes were obtained from the 269 genotypes and a phylogenetic tree was constructed . Further analysis showed that these two wild genotypes have the RLL2 haplotype similar to that of the S1 parent, that is with three copies of RLL2 homologs (RLL2_CGN05092 and RLL2_CGN11333). However, the expression of the RLL2_CGN05092 gene was significantly lower than that of the RLL2 gene in S1. We obtained the genomic sequences of RLL2_CGN05092, including 1.43 kb of sequences in the promoter region. The difference between RLL2 and RLL2_CGN05092 is a 2‐bp (TA) insertion in RLL2 at −411 bp and three SNPs in the coding region. It is most likely that the 2‐bp insertion in the promoter of the RLL2 gene from S1 activates the gene, resulting in red leaves. Therefore, spontaneous mutations in the RLL2 gene might have been selected after domestication  RLL1 is complementary to each other and (ii) homozygotes of loss‐of‐function mutation for either of them completely block anthocyanin biosynthesis in lettuce, which were also the characteristics of the C and T genes  were easily identified through an analysis of allele frequencies in two contrasting pools. Due to several factors, the other two loci (RLL3 and RLL4), which are QTLs, were not detected in our initial analysis. First, the two qualitative loci are epistatic to the two quantitative loci, and consequently, the differences in the allele frequencies of the two quantitative loci were below detection limits. Second, although a large number of individuals (50) in the green pool may reduce sampling error, a large number of individuals in the red pool may decrease the frequencies of the two colour‐promoting quantitative loci. Furthermore, if the original F2 population had been larger, we would have identified the two quantitative loci using only the dark red individuals for the red pool. Furthermore, due to the epistatic effects of RLL1 and RLL2, we needed to use only red individuals rather than the entire F2 population to identify RLL3 and RLL4. Since the biosynthetic pathway and the regulatory network that drives the accumulation of anthocyanins are highly conserved in plants, homology searches in combination with expression profiling analysis are commonly used to identify genes that are associated with the accumulation of flavonoids in non‐model species, such as lettuce . These mutations considerably increased the abundance of anthocyanins in several cultivars.Negative regulators of anthocyanin biosynthesis have been reported in plants, such as the RLL4 in anthocyanin biosynthesis in lettuce is highly similar to that of RUP1 and RUP2 in Arabidopsis, since their loss‐of‐function mutations up‐regulate the expression of anthocyanin‐associated genes. However, in contrast to Arabidopsis, RLL4 in lettuce does not regulate anthocyanin biosynthesis through HY5. Our Y2H experiments suggested no interactions between RLL4 and RLL2, therefore ruling out RLL4 as a competitor of TTG1 (the WD40 component of the MBW complex). Nevertheless, anthocyanin biosynthesis in lettuce is UV‐B dependent, and UV‐B treatment induces the expression of HY5. Therefore, it is most likely that HY5 also bridges the UV‐B pathway and anthocyanin biosynthesis pathway in lettuce. Knockout of genes in the UV‐B pathway, such as HY5 and UVR8, in future studies, may shed light on the regulatory mechanism of RLL4 on anthocyanin biosynthesis.The role of bHLH‐encoding gene. On the other hand, some lettuce cultivars develop red leaves when they are grown in optimal conditions. The constitutive up‐regulation of anthocyanin biosynthesis is due to a gain‐of‐function mutation in a gene encoding a MYB transcription factor and loss‐of‐function mutations in two negative regulators. Therefore, lettuce cultivars that develop green leaves and cultivars that develop red leaves were both selected during domestication and in modern breeding programmes, showing typical artificial disruptive selection, that is selection favouring extreme values over intermediate values  with the mutated ans gene is predicted to generate a green lettuce cultivar, which accumulates abundant colourless flavonoids, providing health benefits to consumers.As a salad vegetable, it is important for lettuce to develop diverse colours. Although anthocyanins are appreciated for their health‐promoting benefits, green lettuce is still the main type of lettuce sold on the market. The combination of the anthocyanin‐promoting alleles of four genes  on the campus of Huazhong Agricultural University in Wuhan, China.To study the genetics underlying leaf colour in lettuce, a loose‐leaf cultivar with red leaves Figure  was croset al., For each segregating population, two pools each containing 50 individuals with contrasting phenotypes were constructed. Tissue from the young leaves of 1‐month‐old plants was pooled, and total RNA was extracted using the TRIzol reagent . RNA was quantified and assessed using a Qubit Fluorometer and a Nanodrop spectrophotometer . Paired‐end sequencing was carried out on an Illumina HiSeq 2500 instrument. Raw RNA‐seq data were mapped to the lettuce genome assembly v8 (Reyes‐Chin‐Wo http://www.clontech.com/). The transformed yeast strains were grown on SD/‐Trp/‐Leu medium, and then, different dilutions were spotted on SD/‐Trp/‐Leu/‐His medium in the presence or absence of 45 mm 3‐aminotriazole . The plates were incubated for 3 days at 28 °C. Cell growth was used to assay the activity of the reporter gene.To perform Y1H assays, the oligonucleotide sequences containing the core elements of the bHLH binding region (G‐box and E‐box) and MYB binding site (MBS) were arrayed in tandem and cloned into pHIS2, respectively. The coding region of the transcription factors was cloned into pGADT7. The yeast strain AH109 was transformed with each construct using the LiAc‐PEG method as described in the manual from the manufacturer , arrayed in tandem and cloned into pHIS2 vectors. Three truncated fragments of the RLL1 coding sequence were fused to the AD domain coding sequence to test whether RLL1 can interact with the promoter sequences of CHS, CHI, DFR, F3H and ANS because the RLL1 protein had a strong autoactivation activity when fused to the GAL4 activation domain.To test whether the RLL1 transcription factor binds the promoters of the genes encoding anthocyanin biosynthetic enzymes, the oligonucleotide sequences containing the core elements of the bHLH binding regions, the E‐box (CANNTG) and the G‐box (CACGTG) in the promoters of the anthocyanin structural genes were identified at PlantCARE . The transformants were selected on SD/‐Leu/‐Trp medium. The positive colonies were transferred to SD/‐Leu/‐Trp/‐His/‐Ade medium containing X‐α‐Gal  to test for interactions between pairs of proteins. Photographs were taken after 3–5 days of growth on the medium at 28 °C.Y2H analysis was performed using the Matchmaker Gold Yeast Two‐Hybrid Library Screening System (Clontech). The ORF of Agrobacterium tumefaciens strain GV3101 was transformed with the constructs that were used for plant transformation using the freeze–thaw method. Transgenic plants were generated using cotyledon explants .To transform lettuce, the full‐length cDNAs from genes of interest were amplified and cloned into pHellsgate8 (Invitrogen), pRI101 and p06 vectors driven by CaMV 35S promoter. 3OH containing 1% HCl) at 4 °C for 16 h. The extracts were centrifuged at 1878 g at 4 °C for 10 min. The supernatants were transferred to new 2‐mL centrifuge tubes. The extracts were filtered using 0.22‐μm nylon syringe filters before being analysed by LC‐MS. Gradient elution was performed at a flow rate of 0.3 mL/min with solvent A (1% aqueous acetic acid) and solvent B (1% acetic acid and 95% acetonitrile) as follows: 0–20 min, 5%–95% B; 20–24 min, 95% B; 24–26 min, 95%–5% B; and 26–30 min, 5% B. The positive ion mode (m/z M+H+) was used to detect the anthocyanins.Lettuce leaves were lyophilized and ground into a fine powder. A total of 0.2 g of this fine powder was extracted with 3 mL of extraction solution . For the last five clones that were sequenced, individual clones were sequenced until no new sequence data were obtained. Sequences were aligned using Geneious . Approximately 2 μg of total RNA per sample was used to synthesize first‐strand cDNA using TransScript One‐Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech). Quantitative RT‐PCR was performed using SYBR premix Ex Taq  and the CFX96 Touch System . The amplification programme was performed at 95 °C for 30 s, followed by 95 °C for 1 s and 60 °C for 10 s (40 cycles). Ubiquitin was used to normalize the qPCR data.RNA‐seq data are available at GenBank under the following accession number PRJNA512330, and gene sequences have accession numbers of MK522155–MK522161.J.C. and H.K. designed the project. W.S., R.T., W.L., C.Y., Z.Y., S.H., D. L. and G.A. performed the experiments. W.Z., L.Z., Y.Z., Q.H. and D. L. analysed the experimental data. J.C., W.S and R.T. wrote the manuscript with the help of H.K., R.M.L. and R.W.M.The authors declare no conflict of interests.Figure S1 Characterization of RLL1.Figure S2 Mapping and functional analysis of the RLL2 gene.Figure S3 Phylogenetic analysis of RLL4 and its homologs.Table S1 Summary of the 145 wild lettuce accessions.Table S2 Primers used in this study.Click here for additional data file.


Phase‐pure oxide nanoparticles with a diameter in the range of 40 to 70 nm were prepared by using an ionic liquid route and deposited onto mesoporous carbon films. The structure and surface composition of the nanoparticles are probed by XRD, TEM, EDX, and XPS. Electrochemical studies carried out under alkaline conditions show a strong correlation between the activity of Lax1‐BaxMnO3 and the effective number of reducible Mn sites at the catalysts layer. Our analysis demonstrates that, beyond controlling particle size and surface elemental segregation, understanding and controlling Mn coordination at the first atomic layer is crucial for increasing the performance of these materials.The electrocatalytic activity of La Indeed, Lee et al. found that Ba substitution resulted in profound changes in the reducibility and availability of surface manganese as evidenced by temperature‐programmed reduction (TPR) and desorption (TPD), which also manifest itself in increased catalytic activity towards CO2 reforming of CH4.x1‐BaxMnO3 nanoparticles with bulk compositions of x=0, 0.15, 0.30 and 1 are synthesised and characterised by XRD, TEM and EDX. The surface composition of the particles was investigated by XPS, while the effective number density of reducible Mn sites was estimated by cyclic voltammetry. The activity of the oxide nanoparticles supported on carbon electrodes towards the ORR was assessed by rotating ring‐disk electrode under alkaline conditions. For the first time, we show that the kinetically limited current is proportional to the square of the number density of reducible Mn sites. The implications of this observation with respect to the ORR mechanism are briefly discussed.A review by Stoerzinger et al. illustrates the wide range of activity reported by Mn‐based oxides, putting into perspective the lack of understanding of the basic principle guiding the reactivity of these materials in comparison to metal catalysts.x1‐BaxMnO3 nanoparticles were synthesised by a highly versatile ionic‐liquid based method which have generated phase pure lanthanides,3)3.6H2 O  and Mn(NO3)2.4H2O  were then added to the ionic liquid and heated under stirring for 2–3 hrs to dehydrate the precursors. After this period microcrystalline cellulose  was added and allowed to stir at 80 °C for 10 minutes, resulting in a homogenous mixture. The resultant mixture was calcined immediately after in an alumina crucible with a ramp rate of 5 °C per minute. All samples were initially heated to 700 °C with a dwell time of 2 h. Lax1‐BaxMnO3 required a secondary annealing process of 5 h of 850 °C and 1100 °C for x=0.15 and 0.3 to achieve phase purity, whereas BaMnO3 was synthesised at 950 °C. The requirement of higher annealing temperatures to incorporate increasing amount of Ba has been previously reported.La−10 mbar. Core level photoemission spectra  were collected in normal emission at room temperature with a non‐monochromatised Al Kα X‐ray source (1253.6 eV) and using 0.1 eV steps, 0.5 s collection time and 20 eV pass energy. The binding energies (BE) were referenced to the C 1s peak at 284.6 eV. The surface composition of the samples was obtained from the Ba 4d, Mn 3p and La 4d peak regions taking into account the corresponding sensitivity factors.X‐ray diffraction (XRD) of the materials was performed with a Bruker AXS D8 Advance diffractometer equipped with a CuKα radiation source. The structures were also investigated using scanning (JEOL SEM 5600 LV) and transmission electron microscopy (JEOL JEM‐1200). Samples for TEM were produced by placing 1 μL drops of the oxide particles dispersed in ethanol on a 3 mm carbon‐coated copper grid. Mean particle diameters were estimated from at least 100 nanoparticles per sample. Photoemission data was obtained in a custom designed UHV system equipped with an EA 125 Omicron electron analyser with five channeltrons, working at a base pressure of 10The EXAFS measurements were carried out at room temperature at the SAMBA beamline of Soleil Synchrotron . The beamline is equipped with a sagittally focusing Si 220 monochromator and two Pd‐coated collimating/focusing mirrors to remove higher harmonics. Calibration of the monochromator was carried out using a Mn foil. Samples were pelletised as disks of 10 mm diameter with 1 mm thickness using cellulose powder as a binder, and XAFS spectra were recorded in transmission mode at the Mn K‐edge (6539 eV). A total of three spectra were averaged for each sample. The spectra were aligned using the Mn foil response. The data was analysed using the Athena software.2) operated with an ALS rotation controller and an Ivium‐CompactStat bipotentiostat. The collection efficiency of the RRDE was determined to be 0.42. All experiments were measured using a Hg/HgO reference electrode (0.1 M KOH) with a graphite counter electrode. Electrodes were prepared via a two‐step drop‐casting procedure leading to a controlled loading 250 μg oxide cm−2, 50 μg Vulcan cm−2 and 50 μg Nafion cm−2.Electrochemical measurements were carried out using a rotating ring‐disk electrode . LaMnO3 was indexed to a cubic (Pm3m) phase, whereas the mixed composition samples were indexed to the rhombohedral space group (R‐3cH) in a hexagonal setting which, is consistent with previous findings by Chakraborty et al.3 was indexed to the P63/mmc hexagonal space group. Quantitative elemental analysis by SEM‐EDX indicates that the bulk elemental stoichiometry is close to the elemental composition of the precursor solution (see Table S1).Figure 2 g−1 (LaMnO3) to 13.9±0.7 m2 g−1 (BaMnO3).TEM images were used to calculate mean particle sizes; representative images are shown in Figure S1 and their particle size histograms can be found in Figure S2. The specific surface areas of the particles (SSA) reported in Table S2 are calculated from the mean particle diameter measured by TEM. The data shows a systematic increase in particle size with increasing Ba content from 33.8±1.6 to 73.5±3.5 nm. This trend leads to a decrease in the SSA from 25.91±1.3 mp, Ba 3d and La 3d regions. The surface composition of the Lax1‐BaxMnO3 nanoparticles shown in Table S3 has been estimated from the area of the Mn 3p, Ba 4d and La 4d peaks and their corresponding sensitivity factors. LaMnO3 and BaMnO3 show A/B ratios at the surface not far from the expected bulk ones. On the other hand, there is significant A‐site atomic surface enrichment in the case of the mixed A‐site samples. This is a clear evidence of a surface segregation of La and Ba forming their corresponding oxides/hydroxides which could be rationalised in terms of the higher temperatures required for generating mixed A‐site samples. XPS data also show a significantly different La/Ba ratio at the surface with respect to the bulk one in the case of La0.70Ba0.3MnO3. This could be a manifestation of the strain relaxation process associated with the bulkier Ba++ cations which can facilitate the surface segregation.Figure 5/2 binding energy (BE) as a function of the Lax1‐BaxMnO3 composition. The mean oxidation state of Mn sites is expected to be +3 in LaMnO3, +4 in BaMnO3 and within +3 and +4 in Lax1‐BaxMnO3 samples. This is substantiated by the X‐ray absorption near‐edge spectra (XANES) at the Mn K‐edge data reported in Figure S3: they clearly show a higher energy for the characteristic pre‐edge feature in the case of BaMnO3 with respect to LaMnO3. Comparing the pre‐edge position with standard Mn‐oxides, following protocols reported elsewhere,3 and BaMnO3 is very close to +3 and +4, respectively. However, the close XPS Mn 2p5/2 binding energy (BE) of Mn3+ (641.9 eV) and Mn4+ (642.2 eV) makes very challenging to quantitatively establish the effective Mn oxidation state at the surface  does not support this assignment.Figure xBa1‐xMnO3 nanoparticles in Ar‐saturated 0.1 M KOH solution at 0.010 V s−1. The current is normalised by the mean oxide surface area, which is estimated from the nanoparticle loading (250 μg cm−2) and the SSA shown in Table S2. LaMnO3 displays two characteristic responses at 0.9 V and 0.5 V, which have been ascribed to the reduction of Mn from the initial oxidation state to a 2+ state.x higher than 0.15. Although a slight variation in the peak current ratio, there is very little difference in the voltammograms of LaMnO3 and La0.85Ba0.15MnO3. This is a somewhat surprising result given that XPS responses reveal a significant A‐site segregation in the case of La0.85Ba0.15MnO3 with respect to the pure lanthanide. Although the trends observed on Mn surface population estimated by electrochemical and photoemission measurements are qualitatively consistent in the case of LaxCa1‐xMnO3,et al. who found a correlation between ORR activity and Mn surface density as determined by cyclic voltammetry but not with the XPS responses.0.70Ba0.3MnO3 and BaMnO3 is rather significant to be exclusively rationalised in terms of surface segregation. Previous studies comparing Sr, Y and Ca manganite perovskites also show a significant influence of the A‐site in the redox Mn responses.Figure  loading 0 μg cm−2Mn) as a function of the bulk Ba content, estimated from integration of the voltammetric responses as exemplified in Figure S4.2+ state from an average initial state calculated from the bulk composition and a ABO3 stoichiometry. We acknowledge that the composition of the first atomic layer, including oxygen content, is expected to be difference from the bulk. However, we anticipate that the dispersion in the electrochemical measurements and in the surface composition in the ensemble of particles is larger than the error incurred in our assumption. Further details of the calculation are summarised in Table S4. To obtain ΓMn, the number of Mn atoms estimated from the charge in the voltammograms is normalised by the specific surface area of the oxides calculated by TEM (Table S2). LaMnO3 and La0.85Ba0.15MnO3 exhibit comparable ΓMn values, whereas La0.70Ba0.15MnO3 and BaMnO3 show a lower value due to the increased Ba content.Figure 2 reduction in 0.1 M KOH at the different catalysts recorded with a rotation rate of 1600 rpm. LaMnO3 and La0.85Ba0.15MnO3 exhibit an ORR onset potential significantly more positive than La0.7Ba0.3MnO3 and BaMnO3. The RRDE responses recorded for the glassy carbon electrode and in the presence of the mesoporous carbon layer under identical conditions are shown in Figure S5. It is interesting to notice that the onset potential for ORR overlaps with the Mn redox signals for all the synthesised samples, as illustrated in Figure S6. This further supports the notion that the ORR to OH− at these oxides is triggered by changes in the redox state of surface Mn sites. It is also observed that the diffusion limiting current is smaller in the case of BaMnO3 and La0.70Ba0.30MnO3, while a larger iRING is observed in the range of 0.2 to 0.65 V. Using the collection coefficient of the RRDE electrode, the effective number of transferred electrons (n) and the hydrogen peroxide yield (% OH2-) were calculated and displayed in Figure S7. The trend in OH2− yield is BaMnO3>La0.70Ba0.30MnO3>La0.85Ba0.15MnO3>LaMnO3, with n values close to 4 for LaMnO3 and La0.85Ba0.15MnO3 while BaMnO3 is close to 3 at potentials around 0.6 V. The observation that BaMnO3 exhibits a mixed selectivity towards both ORR mechanisms is consistent with a previous study by Yang et al.,3 nanorods exhibited a degree of selectivity towards the 2e− mechanism. It should also be mentioned that contributions from the carbon support become significant at potentials more negative than 0.65 V .Figure 3 are closer to that for a 4e− reduction. On the other hand, La0.85Ba0.15MnO3, La0.70Ba0.30MnO3 and BaMnO3 show a change in slope with the increase of rotation rate, implying a combination of the two reactions taking place. In order to minimise the contribution of the carbon support to the kinetic analysis describe below, we shall focus on the electrochemical responses at a potential of 0.65 V. However, we acknowledge that the contribution of the carbon support to the overall current is not entirely negligible at this potential in the case of BaMnO3.Figure S7 shows the Koutecky‐Levich plots for the different electrodes, which display a complex behaviour associated with the interplay between the two and four electron ORR. The slopes observed for LaMnOjk) at 0.65 V normalised by the oxide real surface area as a function of ΓMn. jk is estimated by extrapolating the faradaic current to infinite angular rotation based on Koutecky‐Levich formalism . This representation clearly shows that increasing the number density of reducible Mn sites leads to an increase in the overall activity of the catalysts. Intriguingly, the slope in the logarithmic plot is significantly closer to a value of 2 than 1, suggesting that the rate limiting step in the process is second order with respect to the number density of redox active Mn sites. To the best of our knowledge, such a correlation has not been reported in the context of ORR at non‐metallic catalysts, further demonstrating the importance understanding the chemical nature of the first atomic layer the active sites at oxide nanoparticles.Figure 2 bridges to B‐sites.3 at room temperature,− ORR pathway which is a key defining aspect to Mn based perovskites. Interestingly, DFT calculations by Ahmad et al. concluded that bridge oxygen binding is the most stable adsorption mode at fivefold coordinated Mn sites at the (101) surface.2 binds a single B‐site, but also on the sequence of elementary steps which can lead to the formation of HO2−.Although establishing a detailed mechanistic analysis is beyond the scope of this work, it could be argued that a second‐order dependence on reducible Mn surface sites is more consistent with the so‐called “side‐on” mechanism in which O3x1‐BaxMnO3 (0≤x≤1) nanoparticles towards ORR was assessed as a function of the number density of reducible Mn sites. The nanostructures were characterised by XRD, TEM, EDX and XPS, providing a thorough analysis of their crystal structure, bulk and surface composition. The formation of phase pure mixed La/Ba A‐site perovskites required higher temperatures than in LaMnO3 and BaMnO3. From XPS analysis, temperature above 900 °C promotes a significant segregation of the A‐site to the surface, with Ba segregating more extensively than La. On the other hand, cyclic voltammetry shows a gradual decrease in the effective number density of reducible Mn sites (ΓMn) with increasing Ba bulk content above x>0.15. The ORR activity of the oxide nanoparticles supported at carbon electrodes, as probed by rotating ring‐disk electrodes, shows a significant dependence on the bulk Ba content. The larger activity was obtained for x=0 and 0.15, which is consistent with the larger ΓMn value. Interestingly, the kinetically limited current at 0.65 V shows a square dependence on ΓMn for this family of compounds. This trend suggests that the rate determining step in ORR is second order with respect to the number of redox active Mn sites. Our analysis concludes that beyond controlling particle size and surface composition on these complex materials, it is crucially important to determine and control the coordination of the Mn‐site in the first atomic layer.The electrocatalytic activity of phase pure LaThe 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.


The reaction relies on an unprecedented formal [4+1]‐annulation of these quinone methides with allenoates in the presence of a commercially available chiral phosphine catalyst. The chiral dihydrobenzofurans were obtained as single diastereomers in yields up to 90 % and with enantiomeric ratios up to 95:5.The first highly asymmetric catalytic synthesis of densely functionalized dihydrobenzofurans is reported, which starts from  Given these limitations in catalyst turnover and asymmetric induction, we thought that maybe an alternative and slightly more stable acceptor would be beneficial to address these challenges. Thus, we decided to investigate if this methodology may also be extended to the formal [4+1]‐cyclization of the o‐hydroxy‐containing p‐QMs 3 with allenoates 4.2, more stable acceptor molecule may be better suited to establish a truly catalytic as well as asymmetric protocol, which would then allow for the first enantioselective [4+1]‐annulation of p‐QMs 3 to access the highly functionalized chiral dihydrobenzofurans 5  compoundsa formal +1‐cyclizp‐QM 3 a and diethyl allenoate 4 a in the presence of PPh3 (Table o‐QMs 22 to compensate for its competing decomposition under the reaction conditions). Gratifyingly the targeted dihydrobenzofuran 5 a could be obtained as a single diastereomer in this initial attempt already (entry 1). The relative configuration of the product 5 a was confirmed by NOESY experiments (as shown in Scheme 5 later (Scheme 3 a being recovered (indicating its increased stability compared to o‐QMs 2), we next increased the amount of base (entry 2), which however had a detrimental effect .We started our investigations by carrying out the racemic reaction between 3 a was not very fast in the first attempts with 2 equivalents of base, we next used an excess of allenoate 4 a, which led to a measurable increase in yield (entry 3). The screening of different solvents revealed that toluene allows for a slightly higher yield (entry 4), but also accompanied with a more pronounced formation of various not identified side‐ or decomposition products. Other solvents did not give satisfactory results (see entry 5 for one example) and so further optimizations with CH2Cl2 were carried out. Very interestingly, lowering the amount of base (entry 6) significantly improved the yield and suppressed side product formation. By testing other bases, K2CO3 turned out to be the most promising (entry 7). It should be noted that other simple trialkylphosphines were tested as well,As decomposition of the acceptor 3. Gratifyingly, and in sharp contrast to the reaction with o‐QMs 2,3 allowed for the same yield as when using a stoichiometric amount (compare entries 7 and 8). Further lowering of the catalyst amount unfortunately slowed down the reaction measurably (entry 9). Considering the beneficial effect of using less base when using Cs2CO3 (entry 6), we finally also lowered the amount of K2CO3 , and much to our surprise the reaction proceeded well even without any base .With these first high yielding conditions set, we next lowered the amount of PPh5 a we next focused on the use of chiral phosphine catalysts. As already mentioned before, we were not able to identify a suited asymmetric catalyst for our previous [4+1]‐annulation of o‐QMs 2. However, given the fact that p‐QM 3 a performed very well in the racemic reaction and also allowed for a catalytic approach, we were confident that the well‐described bulky chiral phosphines ABA1–3, but unfortunately neither of them allowed for any product formation (entries 1–3). Gratifyingly however, by switching to the commercially available chiral spiro phosphine B ((R)‐SITCP)2Cl2 (entry 4). Lowering the reaction temperature to 0 °C unfortunately did not allow for product formation anymore (entry 5). When carrying out the reaction in the presence of two equivalents of K2CO3, the outcome was only slightly affected in this solvent (entry 6).Having established high yielding and robust catalytic procedures for the racemic synthesis of 2CO3 as well as under base‐free conditions performed very similarly, with a slightly higher e.r. in the absence of base (compare entries 7 and 8). However, when we further investigated the application scope, we realized that the base‐mediated conditions were more robust when using differently substituted starting materials 3, while not all of those allowed for good conversions under base‐free conditions. Other bases were found to be less satisfactory . We thus tested if any further improvement in the presence of 2 equivalents of K2CO3 may be possible (entries 8–11). However, lowering of the reaction temperature was possible to some extent only , but reducing the catalyst loading to 10 mol % was unfortunately not possible anymore (entry 11). Accordingly, the best‐suited and most robust catalytic enantioselective approach to access 5 a as a single diastereomer was to carry out the reaction in toluene at 10 °C in the presence of 2 equivalents of K2CO3 by using 20 mol % of the commercially available phosphine catalyst B .Interestingly, when changing to toluene (other solvents like THF were found to be not suited), we were able to improve the enantioselectivity significantly (entries 7–9). At room temperature reactions in the presence of K5 a, we next tested the use of differently substituted quinone methides 3 and allenoates 4 . Then it turned out that a dimethyl‐based p‐QM 3 can be used as well to obtain the enantioenriched product 5 c . Interestingly, substituents in the 5 and 6‐position of the benzofuran backbone were very well tolerated . In contrast, substituents in positions 4 and 8 turned out to be more limiting and product 5 g was only accessible with a rather low enantiomeric ratio of 79:21. Surprisingly, compound 5 h was not formed at all under the asymmetric conditions (even with longer reaction times). We were however able to obtain racemic 5 h in high yield when using PPh3 as an achiral catalyst. Very interestingly, while we found initially that benzyl ester containing allenoates were tolerated similarly well as ethyl ester‐based ones (see targets 5 a and 5 b), we found that tert‐butyl esters resulted in somewhat lower enantiomeric ratios compared to ethyl and benzyl esters . All asymmetric reactions were initially carried out on 0.05 mmol scale of the limiting agent 3 and we also reproduced selected reactions on up to 0.2 mmol scale without affecting the outcome, thus indicating that the asymmetric procedure is of similar robustness as the racemic one ‐enantiomer as the major product, but unfortunately, it has not been possible to obtain suitable crystals of any of the products tert‐butyl groups of the phenol derivatives obtained by addition of nucleophiles to QMs can be cleaved off under (Lewis) acidic conditions.5  and which could also be isolated after column chromatography (NMR clearly confirmed that the ester group on the stereogenic center was hydrolyzed). It should be noted that no further attempts to optimize this reaction were undertaken, but this result clearly shows that the highly functionalized compounds 5 a can be used for further transformations and that the two ester groups have different reactivities.It has been described by others that the 5 Scheme . Carryino‐QMs 2 where we found that intramolecular rapid proton transfers are crucial to explain the outcome of this [4+1]‐cyclization.Mechanistically this is a rather complex reaction and it should be admitted that so far, we only have some hints that may allow us to postulate the mechanistic scenario depicted in Scheme I after proton transfer on the primary addition product. Following the reaction between PPh3 and 4 a by 31P NMR shows the appearance of two new signals around 27 ppm (the parent PPh3 peak is at −5 ppm) substantiating the formation of alkylated phosphine species (these addition products decomposed very quickly in the absence of any electrophile). Upon addition of the quinone methide 3 a immediately a strong red color evolves, which can be rationalized by the 1,6‐addition of I to 3 a to give the phenolate II. Similar color changes can also be observed when adding different nucleophiles to other p‐QMs, substantiating the assumed initial 1,6‐addition. With respect to the nature of the electrophile 3 a one could however also postulate that a prototropic shift from the phenol to the para‐QM moiety gives an ortho‐QM in situ, which then reacts with I to give III directly.3 a being rather stable under basic conditions and we never observed any other species or got any experimental hint that supports this pathway, but it should not be ruled out completely with the current state of knowledge. The phenolate II then needs to undergo two proton transfer reactions towards the betaine IV, which can then finally react to the product 5 a via an Sn2′‐type cyclization. We have recently shown for the cyclizations of o‐QMs 2 that these proton transfers are rather likely processes and we reason that the presence of a base is beneficial for these reactions, which would be an explanation why the herein presented [4+1]‐cyclization is more robust under basic conditions. This beneficial effect of base became especially pronounced in those cases where no electron‐donating ring substituent para to the OH group is present . This observation supports a scenario where the final ring closure may be the rate‐determining step, which also rationalizes why slightly larger amounts of catalyst were necessary to obtain satisfying catalyst turnover.Addition of the phosphine to the allenoate is supposed to give the required zwitterion II or III. However, as the observed enantioselectivity was more or less the same under basic and base‐free conditions , K2CO3 (2 equiv), and chiral phosphine B (20 mol %) was cooled to 10 °C and a solution of the allenoate 4 (2 equiv) in dry toluene (20 mL per mmol 4) was added. The resulting mixture was stirred at 10 °C under an Ar atmosphere for approximately 20 h. The mixture was diluted by adding CH2Cl2 (5 mL), filtrated over a pad of Na2SO4 and the residue was rinsed with CH2Cl2 (5×5 mL). The combined organic layers were evaporated to dryness (under reduce pressure) and the products were purified by silica gel column chromatography (gradient of heptanes and EtOAc) giving the corresponding dihydrobenzofurans 5 in the reported yields and enantiopurities .A mixture of the αD23=64.6 ; 1H NMR : δ=0.82 , 1.36 , 1.36 , 1.89 , 3.52–3.77 , 4.26–4.36 , 5.11 , 5.24 , 6.40 , 6.83 , 6.94 , 7.01–7.09 , 7.19–7.26 ppm . 13C NMR : δ=13.4, 14.2, 15.5, 30.2, 34.2, 55.8, 60.8, 61.1, 94.0, 110.2, 121.8, 125.7, 126.1, 128.9, 129.4, 130.0, 132.7, 133.2, 135.1, 153.0, 158.1, 167.2, 168.4 ppm; HRMS (ESI): m/z calcd for C31H40O6: 509.2898 [M+H]+; found: 509.2897. The enantioselectivity was determined by HPLC .Obtained as a yellow residue in 89 % and e.r.=94:6. 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.