Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate‐dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments. Arabidopsis roots adjust their growth to the type of nitrogen source via phosphorylation of auxin carrier PIN2 and adjustment of auxin distribution between neighboring tissues. The ability to sense and adapt to fluctuations in nutrient availability is essential for the survival of all organisms. Every life form on our planet possesses delicate mechanisms for sensing and reacting to the variable nutrient status and adjusts their behavior to maintain growth or cope with stress caused by malnutrition. Mineral nutrients absorbed from the soil are major determinants of plant growth and development. Although required, fluctuations in their availabilities either to sub‐ or supra‐optimal levels often have detrimental effects on plant metabolism and physiology, thereby attenuating plant fitness. Hence, the acquisition of mineral nutrients from the soil needs to be tightly controlled and endogenous levels within a plant body maintained at a physiological optimum level. At the molecular level, balancing nutrient acquisition with the plant’s requirements implies that there is close communication between pathways controlling uptake, distribution and homeostasis of nutrients, and the pathways coordinating plant growth and development.et al, et al, et al, The root system perceives and integrates local and systemic signals on the nutrient status to regulate activity of pathways mediating nutrient uptake and distribution. An important component of the plant’s nutrient management strategy involves a rapid modulation of the root growth and development. In response to nutrient availability, root meristem activity and elongation growth of primary root, as well as root branching, are adjusted in order to optimize nutrient provision to the plant body (López‐Bucio NO3-), nitrite (NO2-), and ammonium (NH4+) as major sources of inorganic N, their preference for different inorganic forms depends on plant adaptation to soil , PIN2, PIN4, and PIN7 and molecular components, which control their subcellular trafficking  was identified as a N responsive gene in the pericycle  is a key macronutrient present in many key biological molecules and therefore constitutes a limiting factor in agricultural systems  and afterward transferred on media containing either NH4+ or NO3-. We found that replacement of NH4+ by NO3- rapidly enhanced root length and already 6 h after transfer (HAT), roots were significantly longer compared to those supplied with NH4+  was enhanced, presumably as a response to stress caused by transfer of seedlings to a fresh plate. Within ~120 min, RGR stabilized at an average speed of 1.37 ± 0.025 µm/min. Transition to dark period correlated with a rapid drop of RGR to 0.98 ± 0.029 µm/min, which was maintained during the dark phase and at the light recovered again to 1.27 ± 0.048 µm/min. Seedlings transferred to NO3- reacted by an increase of RGR to 1.77 ± 0.042 µm/min and similarly to roots on NH4+, during the dark period their RGR decelerated and was retrieved to 1.81 ± 0.051 µm/min at the light . The TZ is located between the root apical meristem and elongation zone, and cells while passing this developmental zone undergo essential modifications associated with their transition from the proliferative to the elongation phase ) in roots supplied with NH4+  revealed a higher number of mitotic events in cortex compared to epidermal cells in roots transferred to NH4+. On NO3-, similar frequency of cell divisions in both epidermal and cortex cell files was observed  to either NH4+ or NO3- revealed enhanced reporter expression and overall enlargement of the meristematic zone in roots supplemented with NO3-  and mDII‐Tomato (red) fluorescent signals indicated increased levels of auxin activity in the central cylinder of roots in response to replacement of NH4+ by NO3- , while in roots supplied with NO3- the auxin activity profiles followed similar trends of steady increase in both cortex and epidermal cell files  were performed. Six hours after applying 3H‐IAA to the root tip, radioactivity in the proximal zone of the primary roots supplied with NH4+ was significantly lower when compared to roots on either NO3- supplemented or standard Murashige and Skoog (MS) medium  machinery, composed of AUX/LAX influx and PIN efflux carriers, directs the flow of auxin from the shoot acropetally through the stele toward the root tip; from where it is via epidermis basipetally redistributed to the elongation zone. At the TZ, auxin might be redirected from the basipetal stream across the cortex, endodermis, and pericycle back to the stele and the root tip, thereby fine‐tuning levels of auxin at the TZ  was detected in roots supplied with NH4+, whereas in roots on NO3-, auxin activity both in epidermis and cortex followed similar trends , and subcellular trafficking in roots supplied with ions Fig . Likewisurce Fig . InteresNH4+ Fig . Furtheret al, NH4+ when compared to NO3- supplied roots, the ratio between intracellular vs. PM‐located PIN2‐GFP was shifted in favor of intracellular localization and frequently endosomal vesicles with PIN2‐GFP signal could be detected  vs. the newly synthesized PIN2‐Dendra  in real time, we could evaluate the kinetics of PIN2 internalization from the PM and delivery of the de novo synthesized PIN2‐Dendra proteins. We found that the kinetics of the photo‐converted PIN2‐Dendra  at the PM in either NH4+ or NO3- were not statistically different, indicating that the internalization of PIN2 is not affected by the N source. Nevertheless, recovery of the newly synthesized PIN2‐Dendra  was significantly enhanced in NO3- when compared to NH4+ supplied roots  analyses on the apical membrane of the cell. Lateral diffusion of PIN proteins at the PM is negligible . In roots supplemented with either NO3- Fig .NO3- supplied roots, trafficking of PIN2 to the PM and the lateral domains is more promoted when compared to NH4+ supplemented roots.In summary, these results suggest that PIN2 subcellular trafficking, and in particular, the delivery of PIN2 to the PM is differentially adjusted according to the N source. In et al, NH4+or NO3- as their N source revealed that PIN2 was among the proteins exhibiting an altered pattern of phosphorylation in response to NH4+   ; and T101D phosphomimetic‐mutant allele (defective in the low‐affinity NO3- uptake)  Heynh plants were used in this work. The transgenic lines W131Y  medium—boric acid 6.2 mg/l, calcium chloride (anhydrous) 332.2 mg/l, cobalt chloride (6H2O) 0.025 mg/l, cupric sulfate (5H2O) 0.025 mg/l, Na2EDTA (2H2O) 37.26 mg/l, ferrous sulfate (7H2O) 27.8 mg/l, magnesium sulfate (anhydrous) 180.7 mg/l, molybdic acid  0.25 mg/l, potassium iodide 0.83 mg/l, potassium phosphate  170 mg/l, zinc sulfate (7H2O) 8.6 mg/l—which contained 0.5 mM Ammonium Succinate (Santa Cruz Biotechnology) (76 mg/l) as a nitrogen source and supplemented with 0.1% sucrose and 1% agar , pH = 5.8. The nitrate amended media contained 5 mM potassium nitrate (505 mg/l) instead of 0.5 mM ammonium succinate. Seeds were stratified at least for 3 days and grown for 4–14 days at 21°C in a 16 h light/8 h dark cycle.Seeds of Seven‐day‐old light‐grown seedlings were transferred to either ammonium or nitrate amended plates and scanned on a daily basis for 7 days on an Epson Perfection V700 flatbed scanner. Root growth (root length changes over a given period of time) and root length were measured manually using Fiji (v1.52).Cell elongation was measured after 12 h exposure to either to ammonium or nitrate manually with the software Fiji (v1.52).PIN2::PIN2‐GFP, PIN2::PIN2S439A‐GFP, PIN2::PIN2S439D‐GFP, Col‐0, and eir1‐4 roots were used, and the length of each cell in different cell files (epidermis and cortex) was measured manually using Fiji (v1.52).For cell length analysis, confocal microscopic images of propidium iodide‐stained et al, 5 DAG seedlings were mounted on a slice of MS medium—containing either 0.5 mM ammonium or 5mM nitrate—placed into a chambered coverslip (Lab‐Tek) and imaged with Zeiss LSM700, LSM800, or LSM880 inverted confocal microscopes equipped either with a 20×/0.8 Plan‐Apochromat M27 objective or a 40× Plan‐Apochromat water immersion objective. Fluorescence signals for GFP , YFP , PI , and DAPI  were detected. A LaVision 2‐Photon Inverted TriM Scope II from LaVision Biotec with a FLIM X16 TCSPC detector from LaVision Biotec equipped with a Olympus UApo N340 40xW, NA 1.15 was also used. Roots were observed 12 h after transfer to ammonium or nitrate supplemented media. Long time‐lapse imaging was performed using a vertically oriented LSM700 microscope as described previously , maximum intensity projections of confocal pictures were used. Images were handled and analyzed with Fiji (v1.52) and Adobe Photoshop (Adobe Creative Cloud).et al, PIN2‐Dendra into its red form induced by illuminating the region of interest with UV light and the depletion of the red and re‐appearance of the green signals in ammonium or nitrate transferred Arabidopsis roots was followed over time using a vertically oriented LSM700 microscope. Observation of roots initiated 10–20 min after transfer and images was recorded every 20 min (9 stacks/root/recording). The experiment was repeated three times, and each experiment consisted of imaging six roots per condition. Image analysis was performed using Fiji (v1.52). Red and green fluorescent signal changes were measured on 10–10 individual cell membranes in the TZ over a period of 6 h. FRAP experiments were performed as described previously .PIN2‐DENDRA experiments were executed as previously described . Images were obtained using an LSM800 microscope.For PIN2 immunostaining, 5 DAG R2D2 combines RPS5A‐driven DII  from Arabidopsis) fused to n3×Venus and RPS5A‐driven mDII fused to ntdTomato on a single transgene  first, we positioned a segmented line over the nuclei in the corresponding tissues with the ROI manager tool of the software Fiji (v1.52) . Next, we analyzed the fluorescence plot profiles of the different lines with the peak analyzer function of the software Origin (OriginLab Corporation) to find local maxima along the lines, which represented the fluorescence value of the nuclei in the tissues. In case of R2D2, auxin distribution plots were derived by reciprocal mean values of the normalized n3xVenus/ntdTomato ratio. Relative auxin level data in each cell per tissue were graphed after data interpolation using the Origin built‐in algorithm for smoothing.Arabidopsis seedlings, which were incubated on either nitrate or ammonium amended medium for 6–8 h, were mounted on to coverslips as previously described by Johnson and Vert  were collected after 1 and 6 HAT and snap freezed in liquid nitrogen. Frozen root tissue was extracted in Reporter Lysis Buffer (Promega), and LUC activity was measured with the Luciferase Assay Reagent (Promega) in a multiwell plate in a Biotek Synergy H1 platereader.Arabidopsis roots was performed according to a previous report  medium with 15 seedlings as one biological replicate, and three replicates per treatment. The [3H]‐IAA  droplets were prepared in MS medium with 1.25% agar and 500 mM [3H]‐IAA (1.45 ml in 10 ml) and were carefully placed on the root meristem (at the very end of the roots). After incubation for 6 h in the dark, the part of the root which was covered by the droplet was cut, and the remaining root parts were collected and ground completely in liquid nitrogen and homogenized in 1 ml scintillation solution . The samples were incubated overnight to allow the radioactivity to evenly diffuse into the whole volume of the scintillation cocktail. Finally, the radioactivity was measured with a scintillation counter (Hidex 300XL), with each sample counted for 100 s, three times. Three samples with only the scintillation solution were used as background controls. As an additional background control, another batch of samples were prepared the same way as described above except [3H]‐IAA containing droplets were placed not on the root meristem but next to the seedlings. Data shown on the figure were calculated against the background.The shootward transport assay of [3H]‐IAA in CycB1::GUS expression was analyzed in seedling roots 7 DAG, 12 HAT to ammonium or nitrate‐containing media. Seedlings were incubated for 2 h in 37°C in staining buffer containing 1 mM ferricyanide, 150 mM sodium phosphate buffer (pH 7), and 1 mg/ml of X‐Gluc dissolved in DMSO. Seedlings were cleared using subsequent incubation at room temperature in a series of ethanol dilutions from 60 to 10% then mounted on slides with 5% ethanol‐50% glycerol mounting solution. The pattern of the GUS histochemical staining was analyzed by an Olympus BX53 microscope and Olympus DP26 digital camera, controlled by cellSense Entry software.® Plant Mini kit (QIAGEN) according to the manufacturer’s protocol. 1 µg of RNA was used to synthesize cDNA using iScriptTM cDNA synthesis kit (Bio‐Rad). The analysis was carried out on a LightCycler 480 II  with the SYBR Green I Master kit (Roche Diagnostics) according to the manufacturer’s instructions. All PCR reactions were carried out with biological and technical triplicates. Expression levels of target genes were quantified by specific primers that were designed using Quant Prime  and then to respective mock treatment.Total RNA was extracted from excised 7 DAG roots 1, 6, and 48 HAT to ammonium or nitrate amended plates using RNeasyt‐test and ANOVA) were performed using the software Origin (v2018). Statistical significance was tested as described in the figure legends.The number of independent repetitions of experiments, as well as exact sample sizes, is described in the figure legends. Statistical analysis (n = 18), tissue , cell position (n = 20) and treatment . The importance of the variables was initially accessed via Random Forest analysis in R (v 1.2.5033). A machine learning training was conducted with the caret R package  programming language and in the L‐system‐based modeling software L‐studio , while z indicates the cell location of the LRC‐derived auxin influx (cell number 20 from the QC). ak represents the rate of active auxin transport between cells via PIN2. The exchange of auxin occurs for each cell j connected to cell i. General processes of auxin degradation like conjugation and oxidation are summarized by a single degradation rate, ad.The ordinary differential equation describing auxin dynamic in a single cell et al, i by approximating functional forms, in what follow:PIN2 is the only auxin efflux carries considered in this model. High auxin concentrations lead to an increased degradation of PIN proteins  or NH4+ (wntr), which in turn is allowed to saturate to zero or to the maximum rate depending on the level of auxin and the distance from the QC, according to logistic coefficients atr and itr, respectively. nl represents the percentage of PIN2 that is redirected to the lateral membranes, depending on nitrate levels. In this model, nitrate level is represented as a binary variable: NO3- for nitrate supplement and 0 for NH4+ supplement.The amount of PIN2 on the membrane et al, i is describes as:v0k denotes the maximal synthesis rate of division factor; ilen and maxL the length of cell i and the maximum cell length achievable, respectively; vt is the tolerance factor restricting the location in the meristem where division takes place; v1k is the level of auxin‐dependent division factor activation. The right part of the formula describes the hypothesized process of division factor degradation, where v2k is the degradation rate of the division factor; v3k and v4k are the level of auxin‐dependent division factor activation and saturation, respectively; 1h and 2h are hill’s coefficient.Cell division is regulated through a hypothetical division factor as proposed in a previous study (Mironova lk indicates the cell elongation rate (depending on whether the cell in the meristem or in the elongation zone), ilen the cell length, and lm the maximum length the cell can achieve (depending whether the cell in the meristem or in the elongation zone).Cell growth is an auxin‐dependent mechanism, and cell entrance in the elongation phase is triggered by an auxin concentration threshold. Both in the meristem and the elongation zone cell growth is defined as follow:et al, et al, Data analysis and plotting were performed using the R language environment for statistical computing  and the plotting package ggplot .Model B: The current model that consider two separate sources from the QC and the LRC (LRC source).Model C: A highly complex model that assume input auxin source modeled as a versatile spline (spline source across epidermis).Model D: A more complex but less realistic model allowing for different independent input of auxin for each epidermis cell (multiple point source).To test auxin source impact on the model predictions, we considered four possible scenarios (Appendix Fig et al, To identify the best model, we tested Models A–D against experimental measurements and generate the information criteria based on the expected log probability density (Appendix Fig Parameters values used in the model are listed below with their estimated mean and lower/upper 95% credible intervals.eir1‐4 background; CC, EBo, and AG designed and performed some of the pre‐pilot experiments; CA assisted KÖ in multiple experiments; JF provided infrastructure, resources, and scientific supervision for AJ, YZ, and ST The manuscript was written by KÖ, KW, and EBe.KÖ and EBe conceived the project; KÖ performed most of the experiments; MM and KW designed the computer model; MM implemented the model and performed computer model simulations; AV, JO, and RAG shared unpublished material; AJ performed the 3D‐SIM experiment; RA performed qPCR and GUS‐staining experiments; LA performed the regression analysis; JCM contributed to the multiphoton microscopy imaging; YZ conducted protein sequence alignments; ST generated the DII and mDII lines in the The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Movie EV3Click here for additional data file.Movie EV4Click here for additional data file.Movie EV5Click here for additional data file.Movie EV6Click here for additional data file.Movie EV7Click here for additional data file.Movie EV8Click here for additional data file.Source Data for Figure 1Click here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.


The synthetic strategy consists of three steps: 1) functionalization, via iridium catalyzed C−H borylation; 2) activation, via fluorination of the generated boronate ester with K[HF2] or [N(nBu4)][HF2]; and 3) expansion, via reaction of the trifluoroborate salts with aryl Grignard reagents. The concept was also shown to be viable for a convergent approach. All but one of the conjugated borane dendrimers exhibit multiple, distinct and reversible reduction potentials, making them potentially interesting materials for applications in molecular accumulators. Based on their photophysical properties, the 1st generation dendrimers exhibit good conjugation over the whole system. However, the conjugation does not increase further upon expansion to the 2nd generation, but the molar extinction coefficients increase linearly with the number of triarylborane subunits, suggesting a potential application as photonic antennas.Using a new divergent approach, conjugated triarylborane dendrimers were synthesized up to the 2 Boron branches out: Conjugated starburst borane dendrimers were synthesized up to the 2nd generation, using to a new, divergent, three‐step approach, exhibiting good substrate tolerance. The dendrimers exhibit multiple, distinct and reversible reduction potentials, making them potential materials for molecular accumulators. Photophysical measurements indicate good conjugation up to the 1st generation. Thus, boranes can be employed as Lewis acids or electron acceptors. Therefore, a plethora of different potential applications have been investigated, including linearπ‐electron delocalization. These systems show promising properties for application such as charge transportThree‐coordinate boranes have attracted considerable interest due to their inherent and interesting properties.In the convergent approach, the dendrimer is built up concentrically from the focal point or core moiety, adding one generation at a time. The drawback of this method is that the number of active centers needed for the expansion, increases exponentially with each generation. This also increases the probability of defects. In the convergent approach, the dendrons are synthesized up to a desired generation, and then attached to the focal point in the last step. The drawback of this strategy is the last step, which needs to be highly efficient in order to warrant the effort of first synthesizing the dendrons. However, the probability of defects is lower using this approach. There are several examples of boron‐containing dendrimers.2). The dendrimers show a red shift of their absorption bands as well as a rise of their molar extinction coefficients and an anodic shift of their first reduction potentials with increasing dendrimer size, indicating good conjugation throughout the system. The reduction events can be attributed to reduction of the boron centers. The boron‐containing dendrimers reported by Müllen and co‐workers contain an electron‐rich arylamine core with BMes2 moieties as the outer periphery  was chosen as the core moiety, as it exhibits a very high degree of stability due to the steric protection of the boron center by six ortho‐methyl groups. In the first step , the xylene moieties were regioselectively borylated para to the boron center via iridium‐catalyzed C−H borylation.2]. This transformation is also generally very efficient. Aryltrifluoroborate salts are highly stable compounds and are widely used in cross‐coupling reactions.3]K salts can be employed as convenient electrophiles for the synthesis of triarylboranes.Our method consists of three steps: functionalization, activation and expansion. Trisborane  denote the substituent para to the boron center on the outermost layer.The nomenclature for the dendrimers was chosen as follows: The divergent synthesis is depicted in Scheme BG0H was synthesized according to a literature procedure and was isolated in a good yield.BG0H was subsequently borylated with [Ir(COD)OMe]2 (COD=cyclooctadiene) as the catalyst precursor, di‐tert‐butylbipyridine (dtbpy) as the ligand, bis(pinacolato)diboron (B2pin2) as the boron source, and THF as the solvent. Then, BG0Bpin was fluorinated using KHF2 in THF/H2O to form the activated core moiety 3KBG0BF. Both BG0Bpin and 3KBG0BF were isolated in very good yields on a multi‐gram scale with a very simple work‐up. However, it was apparent that the solubility of 3KBG0BF in polar organic solvents is quite low. As 3KBG0BF is only slightly soluble in THF, a suspension was reacted with an excess of 2,6‐dimethylphenylmagnesium bromide (9 equiv. instead of 6 equiv.), and BG1H was isolated in 31 % yield. Yields of arylation reactions of the aryl‐BF3K salts were found to be strongly dependent on the substituents on the aryl moiety, as electron withdrawing substituents increase the stability of the trifluoroborate salts whereas electron donating substituents decrease their stability.para to the boron center on the outermost layer, we also synthesized the 1st generation derivatives BG1Me, BG1Br, and 2BG1NMe with methyl, bromide, and dimethylamino substituents, respectively. The syntheses were analogous to that used for the preparation of BG1H, but the respective Grignard reagents were employed. The dendrimers BG1Me, BG1Br, and 2BG1NMe were isolated in moderate to good yields. BG1H was purified without the use of column chromatography simply by precipitation from CHCl3 with EtOH. This procedure did not work as well for BG1Me, BG1Br, and 2BG1NMe; therefore, those products were purified by column chromatography. To synthesize the 2nd generation dendrimer, BG1H was borylated analogously to BG0H, and BG1Bpin was isolated in a very good yield. However, BG1Bpin is almost insoluble in most polar or non‐polar organic solvents, with CHCl3 being the exception. This became problematic for the activation step, and several attempts to synthesize 3KBG1BF in various solvent mixtures , or with the addition of a crown ether (18‐C‐6) or phase transfer catalysts ([N(nBu)4]Cl, [N(nBu)4]Br, [N(nBu)4]OH), were unsuccessful. Finally, exchange of the cation of the fluorination agent from potassium to tetrabutylammonium, allowed the isolation of 3N(nBu)4BG1BF in a very good yield. 3N(nBu)4BG1BF is very soluble in a variety of polar organic solvents  and almost insoluble in water. However, it was unclear what influence the exchange of the cation would have on the expansion step. We have previously proposed that exchanging potassium for lithium increases the reactivity due to the thermodynamically more favorable formation of LiF compared to KF.3N(nBu)4BG1BF was reacted with an excess of 2,6‐dimethylphenylmagnesium bromide (18 equiv. instead of 12 equiv.) in THF. It is important to note that 3N(nBu)4BG1BF does not dissolve very well in THF and forms a viscous oil. However, upon addition of the Grignard reagent, a clear solution formed. After purification by column chromatography, BG2H was isolated in a moderate yield, which can be attributed to the fact that BG2H decomposes slowly on silica gel. Unfortunately, we have not yet found a better procedure to obtain pure BG2H.The core moiety 1H and 13C NMR spectroscopy. Only one set of resonances for each of the chemically equivalent protons and carbon atoms was observed. No 11B NMR signals corresponding to the three‐coordinate boron center were observed for most dendrimers in solution, due to the quadrupole moment of boron and, thus, inherent signal broadening combined with their low solubilities. As 3KBG0BF, BG1H, and BG1Bpin were synthesized in larger quantities, solid‐state 11B NMR spectra were recorded . While we struggled with the synthesis of 3N(nBu)4BG1BF and BG2H, we searched for ways to increase the solubility of the precursors. Having previously worked with 2,6‐bis(trifluoromethyl)phenyl (FXyl)‐substituted triarylboranesBG1H with trifluoromethyl groups. Initial attempts to achieve this using the above methodology failed, as the FXyl Grignard reagent exhibits rather low reactivity, and is also unstable at higher temperatures. Using FXylLi was also unsuccessful, as the reaction is very slow, and FXylLi decomposes in THF at room temperature. Instead, we decided to synthesize BFG1H via a convergent approach using the dendron BFD1H that we had previously reported for the synthesis of borane‐containing TADF materials .In contrast, 3 (BG1Bpin) or CH2Cl2 (2BG1NMe and BFG1H) with ethanol (BG1Bpin and 2BG1NMe) or hexane (BFG1H). The molecular structures are shown in Figure Single crystals suitable for X‐ray diffraction were obtained via slow liquid‐liquid diffusion, by layering saturated solutions of the dendrimer in CHCl3 BGBpin or Cst generation dendrimer BG1Bpin molecule exhibits D3 symmetry in the solid state. In contrast, the central boron atoms of the molecules of BFG1H and 2BG1NMe occupy general positions in the crystal structure and, hence, those compounds do not exhibit any symmetry in the solid state. The B−C (1.547(8)–1.609(2) Å) and C−N (1.379(6)–1.446(16) Å) bond lengths are in the expected range BG1Bpin the B−C bonds to aryl rings with ortho‐methyl groups (1.576(3)–1.577(2) Å) are slightly longer than the B−C bonds to aryls without ortho‐methyl groups (1.566(4) Å). Similarly, in BFG1H, the B−C bonds to aryls with ortho‐trifluoromethyl groups (1.602(2)–1.609(2) Å) are longer than the B−C bonds to the inner aryls without methyl groups in the near vicinity (1.554(3)–1.559(2) Å). This is analogous to previously reported ortho‐trifluoromethylaryl substituted triarylboranes.2 groups on the outer aryl rings of 2BG1NMe have an opposite effect on the B−C bond distances, so that the B−C bonds to the inner aryl rings (1.571(7)–1.578(7) Å) are slightly longer than the B−C bonds to the outer NMe2‐bearing aryl rings with ortho‐methyl groups (1.547(8)–1.569(7) Å). We previously observed a similar shortening of the respective B−C bond in the presence of a strong electron‐donating NMe2 group, which indicates a degree of polarization in the ground state.2BG1NMe, the sum of the C−N−C angles around the nitrogen atoms varies from 350.4(5) to 360.0(5)°. In all of these compounds, we observe an effect of aryl ortho‐methylation on the torsion angles of the aryl moieties with respect to the BC3 plane.ortho‐methylated aryls (45.06(14)–54.31(14)°) compared to non‐methylated aryls (22.94(19)–44.39(15)°). In BFG1H, several B⋅⋅⋅F distances in the range 2.584(2)–2.954(2) Å are observed, which are significantly shorter than the sum of the van der Waals radii for boron and fluorine (3.39 Å).ortho‐CF3 aryl moieties.z‐orbital of the boron center, which results in a stabilizing effect of the ortho‐CF3 groups on the triarylborane. The packing of our molecules in their crystal structures is determined by their bulk. Due to the large torsion angles between the aryl moieties and the BC3 planes, there are no π⋅⋅⋅π stacking interactions present between the molecules. The crystal structure of BG1Bpin contains strongly disordered ethanol solvent molecules and, hence, there is no obvious direct interaction between the molecules of the main compound themselves. The crystal structure of BFG1H mainly exhibits H⋅⋅⋅F, H⋅⋅⋅H, and F⋅⋅⋅F contacts, while in 2BG1NMe, H⋅⋅⋅H contacts dominate, and weak interactions are observed involving a disordered dichloromethane solvent molecule of low‐occupation.The borylated 1nBu4N][PF6] as the electrolyte and a scan rate of 250 mVs−1 .Cyclic voltammograms of the six borane dendrimers were recorded in THF with , ϵ(BG1H)=35×103 [M−1 cm−1]). Functionalization at the para‐position of the outer layer slightly increases the molar extinction coefficient (ϵ(BG1Bpin)=47×103 [M−1 cm−1], ϵ(BG1Me)=40×103 [M−1 cm−1], ϵ(BG1Br)=48×103 [M−1 cm−1]), while expanding the system by one more generation greatly increases the molar extinction coefficient (BG2H=114×103 [M−1 cm−1]). Comparison of the extinction coefficients of BG0H, BG1H, and BG2H, shows an almost linear increase with the number of triarylborane subunits. Combined with the fact that no further red‐shift is observed from BG1H to BG2H, this indicates that the maximum conjugation is reached at the first generation for these systems. Thus, the 2nd generation dendrimer BG2H consists of 2.5 localized, conjugated 1st generation subunits that only weakly interact with each other. This phenomenon is of potential application for a photonic antenna.FXyl groups slightly red shifts the absorption and emission and increases the molar extinction coefficient slightly compared to BG1H (ϵ(BFG1H)=45×103 [M−1 cm−1]). This small bathochromic shift is due to the electron‐withdrawing nature of the FXyl moiety. It is surprising that no analogous bathochromic shift was observed for BG1Br, as the electrochemical measurements indicate a very similar electron withdrawing effect for BFG1H. Adding a para‐NMe2 group, which is a strong π‐donor, drastically shifts both absorption and emission bathochromically, and increases the molar extinction coefficient of the lowest energy absorption compared to that of BG1H =60×103 [M−1 cm−1]). Furthermore, a bathochromic shift of the emission with increasing solvent polarity was observed for 2BG1NMe  = 560 nm; λem,max(CHCl3) = 593 nm), but it was fairly small. This indicates that the nature of the transition is significantly changed by the introduction of a strong π‐donor, most likely from a local excitation (LE) to an intramolecular charge transfer (ICT). The small bathochromic shift of about 1000 cm−1 can be attributed to the radial structure of the dendrimers. As such, the change in dipole moment and the subsequent dependence of the emission on solvent polarity can only occur due to symmetry breaking.BG1Br, exhibit mono‐exponential radiative decays of their excited states. Fluorescent lifetimes τf of all of the conjugated borane dendrimers are ca. 2 ns, with BG1Br exhibiting a shorter lifetime τf=1 ns (average over three lifetimes) and BFG1H exhibiting a slightly longer lifetime τf=4.6 ns. We did not measure the lifetime of 2BG1NMe as partial photodecomposition was observed during the measurements (faster in CHCl3 than in toluene). The quantum yields of all compounds are modest (Φf≈0.1), which can be attributed to their fast, non‐radiative decay rates. This is likely due to the large number of degrees of freedom the systems have because of their size and flexibility.Compared to the core moiety 3 symmetry  or D3 symmetry (BG2H), in order to reduce the cost of the calculations. The HOMO and LUMO energies are plotted in Figure DFT and TD‐DFT calculations were carried out to gain further insight into the electronic and photophysical properties of the conjugated borane dendrimers. Due to the large size of the systems, and their apparent high symmetry observed in solution by NMR spectroscopy, all dendrimers were calculated with CBG1Br<BFG1H<BG2H<BG1H<BG1Me<2BG1NMe. The calculated HOMO energies show almost the same trend, except that the order of BG1H and BG2H is switched. The experimental HOMO and LUMO energies also show the same trend; however, the order of BG1Br and BFG1H is switched for the LUMO energies. The calculations describe the trends in HOMO and LUMO energies of the conjugated borane dendrimers rather well, accounting for the fact that all calculations were carried out using symmetry. TD‐DFT calculations were carried out in order to gain more insight into the photophysical properties of the dendrimers. The lowest energy transitions S1←S0 and S2←S0 are degenerate for all dendrimers, due to the symmetry used in the calculations. The natural transition orbitals (NTOs) for the S1←S0 and S2←S0 transitions of all dendrimers are depicted in Figure At the B3LYP/6‐31+G(d) level of theory, the calculated LUMO energy levels rise from BG1Br, BFG1H, BG2H, BG1H, and BG1Me, the S1←S0 and S2←S0 transitions correspond to π(xylene)‐pz(boron) transitions. The outer xylene moieties have a smaller contribution than the inner xylene moieties. In all cases, the virtual NTOs correspond to the LUMO of the respective dendrimer, which are mainly localized on the four boron centers of the core and the 1st generation. This is also the case for BG2H, illustrating the limited conjugation of the 2nd generation dendrimer, and it explains the similarity of its absorption and emission spectra to those of the 1st generation dendrimers. For 2BG1NMe, the S1←S0 and S2←S0 transitions can be classified as charge transfer (CT) transitions from the 4‐dimethylaminoxylene moieties of the outer layer to the four boron centers. This is also supported by the small orbital overlap coefficients ,Λ=0.25 and 0.28) than those of the other dendrimers (Λ≈0.5). The specific orbitals contributing to the transitions vary, depending on the substitution patterns of the outer periphery of the dendrimers.For BG1Br, BG1H, BG1Me, 2BG1NMe, and BG2H. The three step divergent approach, consisting of: 1) functionalization, via iridium‐catalyzed C−H borylation; 2) activation, via fluorination of the boronate ester generated with K[HF2] or [N(nBu)4][HF2]; 3) expansion, via reaction of the trifluoroborate salts with an aryl Grignard reagent, works as designed up until the 1st generation. After the 1st generation, solubility issues forced us to modify the synthesis slightly. The 2nd generation dendrimer BG2H was synthesized after exchanging the cation of the fluorination step from potassium to tetra‐n‐butylammonium, in order to improve the solubility in organic solvents. Substrate tolerance is mainly limited by the reactivity of the aryl Grignard reagent. Additionally, we synthesized BFG1H by a convergent approach. All dendrimers were investigated via cyclic voltammetry, UV/Vis absorption and emission spectroscopy, and by DFT and TD‐DFT calculations, in order to gain more insight into their electrochemical and photophysical properties. All dendrimers, except BG1Br, exhibit multiple, distinct and reversible reductions, that can be easily modified via the substitution pattern of the outer periphery of the dendrimers. As such, they might have potential application as molecular accumulators.st generation, the molar extinction coefficient does, which is of possible interest for the design of a photonic antenna.We have designed and applied a new divergent strategy for the synthesis of conjugated borane dendrimers The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


We investigate the substrates and stereoselectivity of the reaction, characterise the catalyst by a range of physicochemical experiments and propose the reaction mechanism based on density functional theory (DFT) calculations. It is found that three key residues  in ATP are important for the efficient catalytic activity and stereocontrol via complexation of the Cu(II) ion. In addition to the potential technological uses, these findings could have general implications for the chemical selection of complex mixtures in prebiotic scenarios.Natural biomolecules have been used extensively as chiral scaffolds that bind/surround metal complexes to achieve stereoselectivity in catalytic reactions. ATP is ubiquitously found in nature as an energy-storing molecule and can complex diverse metal cations. However, in biotic reactions ATP-metal complexes are thought to function mostly as co-substrates undergoing phosphoanhydride bond cleavage reactions rather than participating in catalytic mechanisms. Here, we report that a specific Cu(II)-ATP complex -ATP complex that efficiently catalyses Diels-Alder reactions, and determine ATP residues that are essential for this activity. To precisely characterise the active centres and obtain insight into the reaction mechanisms of ArMs, simple scaffolds of peptides and amino acids have been employed to rationally design artificial metallo-peptides13 and metallo-amino acids21. In recent years, nucleic acids have aroused much interest among chemists for constructing diverse nucleic acid-based ArMs for enantioselective catalysis. Natural double-stranded DNA (dsDNA) was first employed as a chiral scaffold in a supramolecular assembly with an achiral copper(II) complex, realising the chirality transfer from dsDNA to the products22. Since then, many synthetic dsDNAs have been designed to covalently anchor metallic moieties to produce DNA-based ArMs28. These artificial designs enable the fine-tuning of the microenvironment and provide a deeper understanding of the origin of chiral induction. Through their tuneable structures, G-quadruplexes containing 21- to 69-mer nucleotides have been employed to construct G-quadruplex DNA metalloenzymes, which have been successfully applied to several enantioselective transformations and demonstrated to depend largely on the conformation of the non-canonical G-quadruplex structure36. In addition, a short single-stranded 11 nt DNA within a G-triplex structure was shown to bind to copper(II) ions and modestly promote an enantioselective Diels–Alder (D–A) reaction37.Artificial metalloenzymes (ArMs) are usually constructed by embedding metal cofactors into the chiral scaffolds of biological molecules that are used to expand the reaction types and unearth novel functions of the biomolecules. Over the past four decades, protein-based ArMs have been widely investigated to achieve a variety of valuable enantioselective transformations38. Most importantly for the present work, a ribozyme was selected to catalyse a D–A reaction. The Diels–Alderase ribozyme shows a 20,000-fold rate enhancement and relies on the presence of divalent metal ions such as Mg2+ or Mn2+39. In a further work Jaeschke and co-workers developed a 49-mer Diels–Alderase ribozyme that could catalyse the D–A reaction of anthracene derivatives and maleimides with an enantiomeric excess (ee) of up to 95%40. Systematic experiments based on the anthracene derivative dienes and maleimide dienophiles suggest that the stereoselectivity of the reaction is mostly controlled by RNA–diene interactions and the hydrophobic side chain of the dienophile is responsible for RNA binding41. The crystal structure of the Diels–Alderase ribozyme shows that the Mg2+ ion is a structural cofactor that stabilises the wedge shaped pocket and the stereoselectivity is governed by the shape of the catalytic pocket42.In addition to DNA, a large number of ribozymes generated by in vitro selection exhibit catalytic activity with the assistance of metal ions43. Another RNA-based ArMs containing either dsRNA or hairpin RNA exhibited very low enantioselective induction in a D–A reaction compared with the corresponding DNA-based ArMs44. The current nucleic acid-based ArMs always contain several tens to hundreds of nucleotides to achieve chiral scaffolding. In most cases, the precise location of the catalytic metal species is unclear, and high resolution structures of these ArMs are lacking. Recently, our group reported a cyclic dinucleotide (c-di-AMP)-based artificial metalloribozyme that catalyses a Friedel–Crafts reaction with high enantioselectivity45. Furthermore, a phosphine-modified deoxyuridine coordinating a palladium species enables an enantioselective allylic amination46, suggesting that the chirality of the sugar could be transferred to the product. Therefore, the design of nucleic acid-based ArMs with only a few nucleotides as the scaffold appears to be a promising approach for obtaining minimal systems that might be better suited for gaining accurate structural information and providing deeper insights into the reaction mechanisms. ATP is a well-known energy-storing molecule that participates in many processes in living organisms. In most enzyme-catalysed reactions, ATP acts as a co-substrate undergoing phosphoanhydride bond cleavage reactions. However, ATP has been demonstrated to specifically bind metal ions with high affinity and could therefore function as a chiral scaffold participating in enantioselective catalysis mediated by the complexed metal ions.In addition to in vitro-selected ribozymes such as the Diels–Alderase, synthetic double-stranded RNAs (dsRNAs) were shown to interact with copper(II) complexes to form RNA-based ArMs, giving rise to high reactivity yet modest enantioselectivity in a Friedel–Crafts reaction2+ ions to form a Cu(II)-ATP complex (Cu2+·ATP) that efficiently catalyses enantioselective D–A reactions  and cyclopentadiene (2) was employed to test the catalytic performance of ATP–metal ion complexes. The initial attempt using either ATP or a Mg2+·ATP complex resulted in a very low conversion and ee value (Table 3a (exo) of 74% and an ee of 3a (endo) of 65% –ATP complex of Cu2+·ATP. Using other divalent metal ions such as Zn2+, Co2+ and Ni2+ as the metal cofactors resulted in low conversions and ee values  salts and molar ratios of ATP/Cu2+ were screened  were investigated and modest to good stereoselectivities were obtained  on the phenyl moiety, exhibited an enhanced ee in the corresponding reaction (Table 1d) caused a significant decrease in the reactivity and enantioselectivity (Table 1e and 1f), 1e bearing R = (4-Cl)C6H4 reacted with 2 to give a 93% ee in favour of the exo isomer of 3e (Table 1f with R = (4-NO2)C6H4 yielded an 84% ee in favour of the endo isomer of 3f  was conducted at a large scale to test its potential practicability. At a Cu2+·ATP loading of 5 mol%, the product 3a was obtained with an isolated yield of 80% and an ee value of 65% in favour of the endo isomer. Overall, it was demonstrated that ATP and the Cu2+ ion form a potent and practical entity of Cu2+·ATP for enantioselective D–A reactions.To investigate the substrate specificity of the Cuhalcones b–h were halcones b–h were halcones b–h were halcones b–h were halcones b–h were halcones b–h were 2+, the apparent second-order rate constants (kapp) of ATP, Cu2+ and Cu2+·ATP were determined by monitoring the UV-Vis absorption of 1a during the corresponding D–A reactions. Compared with the D–A reaction without a catalyst , that with ATP had a comparable kapp,ATP –ATP complex, where ATP serves as the chiral scaffold and the Cu2+ ions serve as the catalytically active species.To clarify the catalytic roles of ATP and Cu2+ ion to ATP essential for catalysis, ATP was replaced with different ATP analogues in the Cu2+·ATP catalysed benchmark D–A reaction. Compared with ATP with three phosphates as the scaffold, ADP and Cu2+ ions catalysed the D–A reaction with a similar conversion but a sharply decreased ee value of 19%  and ATP remained nearly unchanged during the reaction at pH 5.5 –ATP complex, the apparent binding constant (kb) was determined by a UV titration experiment. The kb value for ATP and Cu2+ ions was estimated to be (3.2 ± 0.1) × 105 M−1 based on the curve fitting  was employed to verify the interaction between ATP and Cu2+ ions. The EPR spectrum of only Cu2+ showed the typical fine structures of paramagnetic Cu2+ ions  was used to detect possible binding sites of Cu2+ ions in ATP. Owing to the paramagnetic property of Cu2+ ions, the addition of Cu2+ ions to ATP causes NMR signal broadening and shifting. When the ATP/Cu2+ ratio was 300:1, the signal of H8 first broadened and shifted downfield relative to that in the 1H NMR of ATP, and further increasing the amount of Cu2+ ions caused the H8 signal to broaden to the baseline and shift downfield  calculations were performed. Based on several proposed Cu2+·ATP models in the literature56, a stable structure of Cu2+·ATP was obtained by a gas-phase calculation in which the Cu2+ ion is bound to the N7 atom of adenine and the β- and γ-phosphate oxygen atoms and accompanying a  hydrogen bond Pγ-O···H-N6  of the optimised Cu2+·ATP structure was 0.3 kcal mol−1 lower than that of a previously described model obtained by a molecular orbital method57 and 8.7 kcal mol−1 lower than that of the Cu2+·ATP model without a hydrogen bond  as an example, the ΔE value of the precursor of 1a-Cu2+·ATP and 2 that yielded 3a (endo) via the attack of the Si face was 9.1 kcal mol−1 lower than that of the precursor for the Re face attack  with the preferred configuration of 1R, 2S, 3S, 4S  were also calculated. The precursor of 3a (Si-exo) was more stable than that of 3a (Re-exo), further suggesting that cyclopentadiene 2 favoured the attack of 1a-Cu2+·ATP from the Si face  was 3.1 kcal mol−1 lower than that of 3a (Si-exo), in accordance with the experimental results  and Cu···O(1a) interactions. Because of the hydrogen bonding between ATP and 2, 2 preferentially attacked the Si face of 1a in the intermediate of 1a-Cu2+·ATP, leading to a relatively stable transition state consisting of Cu2+·ATP, 1a and 2. The transition state of 1a-Cu2+·ATP-2 automatically converted to the intermediate of 3a-Cu2+·ATP, which was accompanied by the breaking of the Cu···O(1a) bond, and the major D–A product 3a (Si-endo) was obtained after release from Cu2+·ATP.To further substantiate the hypothesised fine structure of the catalytically active Cu-N6 Fig. . The relack Fig. . The Si oms Fig. . This re2+ ions is able to catalyse a D–A reaction with significant rate acceleration and high enantioselectivity. The purine structure and the phosphates at the β- and γ- positions are vital factors contributing to the enantioselective activity of the Cu2+·ATP catalyst. Based on control experiments, physicochemical characterisations, and DFT calculations, a fine coordination structure of Cu2+·ATP in which the Cu2+ ion binds to the N7 atom, β-phosphate oxygen atom, and γ-phosphate oxygen atom and an intramolecular hydrogen bond between the 6-amino and γ-phosphate oxygen moieties was proposed. Of the reported metallo-biohybrid catalysts for enantioselective D–A reactions –ATP complex. The proposed fine coordination structure of the complex is able to explain the origin of chiral induction and should facilitate the rational design of further simple but efficient nucleotide-based catalysts.In summary, we discovered that an enantioselective catalyst composed of the single nucleoside triphosphate ATP in complex with Cu58, even providing early access to nucleotides from very simple starting materials59. However, how mirror symmetry breaking in aldol and other reactions occurred is still an open question61. Since aldol reactions are also catalysed by divalent metal ions65, it is possible that nucleotide polyphosphates have played a role in the chemical selection of important metabolites in the abiotic stages of the emergence of life. Experiments investigating the potential of nucleotides to initiate chiral induction in aldol reactions are currently underway in our lab.In addition to its potential use in synthesis approaches, this work suggests that nucleotides could have played a role in the chemical selection of complex mixtures in prebiotic reactions in early evolutionary scenarios. The observation that several of the currently ubiquitous cofactors involved in redox and C–C bond formation reactions  contain adenine nucleosides and nucleotides could indicate a more pronounced role of ATP as ligand in prebiotic reactions. Although the D–A reaction described in this work is not proposed to be highly important in early chemical evolution, aldol reactions are thought to have played a crucial role in the establishment of early metabolic pathways2  were added to an MES buffer solution  in a 10 mL vial to a total volume of 1000 μL. After stirring for 30 min at 4 °C, a thoroughly mixed solution of azachalcone 1a  and freshly distilled cyclopentadiene 2  were immediately added. After the mixture was stirred for 24 h at 4 °C, the aqueous media were extracted by diethyl ether (3 × 2 mL) and flushed through a short gel column (a 5 cm length of glass dropper was filled with the silica gel to a height of ca. 2 cm with some cotton at the bottom). The combined organic layers were removed under reduced pressure and the residue was directly analysed by chiral HPLC using a Daicel Chiralpak ODH column (250 × 4.6 mm) with hexane and isopropanol as the eluents. The conversion of 1a was calculated using the following Eq.  and a freshly prepared aqueous solution of Cu(OTf)1a at 326 nm, followed by the reference66, using UV–Vis spectroscopy at 4 °C. ATP  in an MES buffer  was added to a 2 mL quartz cuvette containing a small magnet and stirred for 10 min, and then an aqueous solution of Cu(OTf)2  was added. After stirring for another 20 min, a fresh solution of 1a  was added. Followed by an immediate addition of 2 , the measurement was started and the cuvette was sealed tightly. The initial rate (Vinit) was determined from the slope of the line fitted to the decrease in the absorption of 1a versus time, and the following Eq.  was determined according to the procedure described in the literature66. The following Eq. ; the 6–311G basis set was used for C, H, O, N and P, whereas LANL2DZ was used for Cu. The kinetic barriers of the non-catalytic reaction and the catalytic reaction with the most stable precursor were evaluated by calculating the single-point energies at the M06-2X-D3/LANL2DZ ~6–311G level. All the structures were verified to be local minima by frequency calculations, whereas all the transition state species had only one imaginary frequency.All calculations of the reactions were performed in the gas-phase with Gaussian 16Supplementary InformationPeer Review File


II4L4 tetrahedral cages prepared from similar building blocks showed contrasting host–guest properties. One cage did not bind guests, whereas the second encapsulated a series of anions, due to electronic and geometric effects. When the building blocks of both cages were present during self‐assembly, a library of five CoIIALxBLx4− cages was formed in a statistical ratio in the absence of guests. Upon incorporation of anions able to interact preferentially with some library members, the products obtained were redistributed in favor of the best anion binders. To quantify the magnitudes of these templation effects, ESI‐MS was used to gauge the effect of each template upon library redistribution.Two Co A statistical library of dynamic tetrahedral cages was formed by self‐assembly of building blocks of similar sizes. ESI‐MS was used to probe the subtle effects of anion binding on the dynamic library formed. Upon the addition of anions able to bind within the library members, an increase or decrease of the proportion of each congener of the library was observed, allowing for information on relative binding affinities to be gained via MS. Molecular recognition is a fundamental process within biological systems. The conformational restructuring which a system undergoes upon the introduction of additional componentsThe outcome of self‐sortingII4L4 tetrahedral cages prepared from trianilines A or B were reported not to encapsulate guest molecules, due to their small cavity sizes.II instead of FeII might yield structures with slightly larger cavities, thus enabling guest uptake, as CoII cages had previously been shown to encapsulate larger guests than their FeII analogs.1 and 2 , 2‐formylpyridine (12 equiv) and Co(NTf2)2 (4 equiv). The 1H NMR spectra of 1 and 2 were consistent with species of overall T symmetry , while ESI‐MS confirmed CoII4L4 stoichiometry . Single crystals of 1(ClO4)8 and 2(ClO4)8 suitable for X‐ray diffraction were obtained by slow vapor diffusion of either Et2O or EtOAc into solutions of 1 or 2 in CH3CN. The crystal structures .1 was found to crystallize without a guest bound inside its cavity, the crystal structure of 2 showed an encapsulated ClO4− anion.The Fepyridine  equiv an−, I−, BF4−, and ClO4− anions, as well as acetonitrile, were found to bind within 2 (see Supporting Information section 4.2). Internal guest binding was indicated by the appearance of a new set of 1H NMR signals upon addition of these anions, consistent with the formation of a host–guest complex in slow exchange on the NMR time scale.2, in the order Br− > I− ≫ BF4− ≥ ClO4− ≫ CH3CN.− added increased, the concentration of host‐guest complex (X−⊂2) reached a plateau at a concentration below 1:1 binding . The analysis described in section 5 in the Supporting Information allowed us to conclude that anion encapsulation was competing with anion‐TBA+ association  and with the encapsulation of CH3CN, in geared equilibrium processes. These three association phenomena were considered together (Supporting Information section 5.3), producing the following association constants in CD3CN: KBr‐=16000±1000 m−1, KI‐=4800±400 m−1, KBF4‐=580±50 m−1, KClO4‐=1100±100 m−1, KCH3CN=0.0072±0.0004 m−1.Br1 when the anions studied were added following cage formation  or when cage 1 was formed in the presence of the anions . Given that 2 encapsulates a variety of anions, we were surprised that host 1 did not encapsulate any of these guests. Calculations of the cages’ internal voids  indicated that the cavities of 1 and 2 were of nearly equal volumes . The cavity in 1 was expected to accommodate Br− and I−, with occupancies of 48 % and 59 %, respectively (Table S2).4−⊂1 and ClO4−⊂1 are predicted to have ≈95 % occupancy, the X‐ray structure of ClO4−⊂2 demonstrated that such high occupancy is possible. Guest binding may be hindered by the lesser degree of pyramidalization in nitrogen‐centered AL than in hydroxymethyl‐centered BL (Supporting Information Section 3.4). In addition, we hypothesize that the p orbitals on four facial nitrogen atoms contribute electron density to the microenvironment within the cavity, thus destabilizing the binding of anionic guests. This hypothesis is supported by molecular orbital and electrostatic potential calculations (Supporting Information section 3.6).No binding was observed within A and B, and the product complexes 1 and 2, we investigated the formation of heteroleptic libraries of CoIIALxBLx4− cages in the presence of guest molecules. The reaction of A and B (1 equiv each), 2‐formylpyridine (6 equiv) and Co(NTf2)2 (2 equiv) in CH3CN led to the formation of a library of cages (LibNTf2) as confirmed by ESI‐MS and 1H NMR . Single‐crystal X‐ray diffraction afforded a structure displaying disorder that was best modeled whereby each face of each cage had a 50 % probability of incorporating a residue of A, and 50 % B.Given the similarities in size and geometry between tritopic amines Lib′NTf2was formed, where structures of composition CoII4ALxBLx4− with 2≤x≤4 were expressed predominantly . Three sets of imine signals, containing six, four, and one resonance, respectively, were observed in the 1H NMR spectrum of Lib′NTf2. Based upon symmetry, these sets of signals were assigned to CoII4AL2BL2, CoII4AL3BL and CoII4AL4, respectively. Each of the 16 signals observed in the imine region of the 1H NMR spectrum of LibNTf2were thus attributed to one of the CoIIALxBLx4− cage congeners .When the reaction was carried with sub‐stoichiometric amounts of 2‐formylpyridine (3 equiv instead of 6 equiv), a different library LibNTf2was examined with 1H NMR and ESI‐MS (Supporting Information section 7.2). Since similar ESI response factors were observed for 1 and 2 , we hypothesized that the heteroleptic CoIIALxBLx4− cages might have similar response factors as well. The integral of the signal for each species might thus reflect its concentration, relative to the others present in solution. No significant difference between the ratio of species in LibNTf2was observed with either analytical method (NMR or ESI‐MS), validating the use of ESI‐MS spectra to determine the relative concentrations of cage species in solution. In addition, the collection of CoII4ALxBLx) were prepared by the addition of the tetrabutylammonium salt of the corresponding anion X− (2 equiv) to LibNTf2, therefore precluding quantification of the species present in solution. Thus, detailed ESI‐MS studies were undertaken.Four libraries of mixed cages  displayed clusters of peaks corresponding to individual CoII4ALxBLx). The distributions thus observed deviated strongly from the near‐binomial distribution observed for LibNTf2, with anion templation favoring the incorporation of BL into the structures. The greatest deviations were observed for the smaller anions . Thus, we suggest the high proportion of 1 to be due to it serving as a ‘sink’ for residual A that were not incorporated into the anion binding cages  should be addressed to the authors.SupplementaryClick here for additional data file.


Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:653–662, 2020One possible approach to treat osteoarthritis (OA) is to counteract cartilage degeneration with anabolic compounds that stimulate chondrocyte proliferation and/or extracellular matrix (ECM) production. Several molecules including sprifermin (recombinant human fibroblast growth factor [FGF18]), insulin‐like growth factor‐1 [IGF1] and ‐2 [IGF2], C‐type natriuretic peptide [CNP], and bone metamorphic protein 7 [BMP7] have been shown to have these characteristics both in vitro and in vivo. However, it is not known how these molecules compare each other regarding their effect on phenotype and stimulation of ECM production in primary chondrocytes. The effects of sprifermin, IGF1, IGF2, CNP, and BMP7 were evaluated on bovine articular chondrocytes, first in monolayer to determine their effective concentrations, and then in three‐dimensional (3D) culture at concentrations of 100 ng/ml for sprifermin; 300 ng/ml for IGF1, IGF2, and BMP7; and 10 nM for CNP. In 3D culture, the effects of a permanent exposure or a cyclic exposure consisting of 24 h incubation per week with the compounds were evaluated. All growth factors increased ECM production and cell proliferation to a similar extent but CNP had almost no effect on bovine chondrocytes. Sprifermin was more effective with cyclic exposure, IGF1, and IGF2 with permanent exposure, and BMP7 showed similar results with both exposures. Regarding the cell phenotype, sprifermin appeared to be the only compound favoring the chondrocyte phenotype; it decreased type I collagen expression and had no hypertrophic effect. Together, these results confirmed that sprifermin is a promising disease‐modifying OA drug. © 2019 The Authors.  Osteoarthritis (OA) is a degenerative joint disease affecting various tissues in the joint, with cartilage degradation being the hallmark of the disease. The associated symptoms include pain and limited functionality of the affected joint, resulting in a considerably reduced quality of life. The most recent Global Burden of Disease report GBD2013) estimated that globally 242 million people are living with symptomatic and activity‐limiting OA of the hip and/or knee.3 estimatThe two main target strategies for potential DMOADs are either anti‐catabolic (which aims to stop the process of cartilage degradation) or pro‐anabolic . Sprifermin, a recombinant human fibroblast growth factor 18 (rhFGF18), is a potential pro‐anabolic DMOAD that is currently in clinical development for knee OA.Several other growth factors have been tested for their anabolic properties on cartilage or chondrocytes. Insulin‐like growth factor 1 (IGF1) and 2 (IGF2) are proteins with a similar structure to insulin. IGF1 has been shown to stimulate cell proliferation and to increase proteoglycan and collagen production in vitro, and to promote cartilage repair in vivo.BMP7, a member of the bone metamorphic proteins (BMPs) is another well‐characterized cartilage anabolic factor. BMP7 has been shown to stimulate the production of proteoglycanFinally, C‐type natriuretic peptide (CNP) is a natriuretic peptide involved in cartilage homeostasis.Alongside their chondrocyte‐anabolic properties, none of these compounds  appear to exert any deleterious effects on other tissues in the joint. This is in contrast to some other agents with chondrocyte‐anabolic properties, such as FGF2, transforming growth factor β (TGF‐β), and BMP2, which were therefore excluded from this study.Escherichia coli and purified as previously described.Sprifermin, a recombinant human FGF18, was expressed in BMP7 was obtained from R&D System , IGF1 and IGF2 were from Merck‐Millipore  and CNP from Sigma‐Aldrich . All protein produced in a cell system had low levels of endotoxin (<0.1EU/µg). CNP was chemically synthesized.Bovine chondrocytes were isolated from the metacarpal joint of cattle aged 1–2 years, collected from a local slaughterhouse. The cartilage was digested in 0.1% (w/v) collagenase NB4  at 37°C overnight and the resulting cell suspension was subsequently filtered, washed, and resuspended in a culture medium with 1% penicillin and streptomycin. The culture medium was composed of Dulbecco's modified Eagle's medium (DMEM) High Glucose media  supplemented with 10% fetal bovine serum , 50 µg/ml ascorbate‐2‐phosphate (Sigma‐Aldrich), and 0.4 mM proline (Sigma‐Aldrich).For the monolayer culture, the cells were first cultured for 1 week in the culture medium. The cells were then harvested and seeded at 15,000 cells per well in a 24‐well plate and cultured in culture medium with 0.3–1,000 ng/ml sprifermin, BMP7, IGF1, and IGF2, or 0.003–10 µM CNP. Two monolayer cultures were performed; one with IGF1, IGF2, and CNP and a second one with BMP7 and sprifermin. After 7 days, the medium was harvested for glycosaminoglycan (GAG) analysis and the cells were detached using accutase (Sigma‐Aldrich) containing 2.5% collagenase. The cells were counted with a Vi‐cell™ XR cell counter  or processed for gene expression.6 cells per well in 96‐well ultra‐low binding plates and cultured for first 1 week in a culture medium without compound and subsequently 4 weeks with IGF1, IGF2, or BMP7 at 300 ng/ml, sprifermin at 100 ng/ml, or CNP at 10 nM. Two different experiments were performed; one with sprifermin, IGF1, and IGF2 and another one with sprifermin, CNP and BMP7. In both experiments, the 3D constructs were incubated with the compounds either permanently or intermittently . At the end of the culture period, the 3D constructs were either used for the analysis of the GAG and hydroxyproline (HPro) content, gene expression, or histological analysis. Prior to biochemical analysis, the constructs were digested overnight at 60°C with papain 0.125 mg/ml  in 0.1 M Na2HPO4, 0.01 M ethylenediaminetetraacetic acid, and 5 mM l‐cysteine. All cultures were done with N = 3–4.For the 3D culture, freshly isolated chondrocytes were used and seeded at 1 × 102H3] from C/D/N‐Isotopes) and 200 µl of 25% (v/v) hydrochloric acid and hydrolyzed overnight at approximately 110°C. After centrifugation, samples were evaporated at 55°C/10 Torr, resuspended in 1 ml water and mixed 1:5 in acetonitrile. The HPLC separation  was achieved on a hydrophilic interaction chromatography column  using a mobile phase gradient . Detection was performed on a tandem MS  with a turbo ion spray interface operating in positive ion mode.GAG was measured with the DMMB (dimethyl methylene blue) assay as previously described.RNA isolation was performed with the RNeasy Mini Kit  according to the recommendation of the manufacturer. The 3D constructs were additionally treated with proteinase K (Qiagen) for 10 min at 55°C before RNA isolation. The messenger RNA (mRNA) concentration and quality were analyzed using an Agilent Bioanalyzer  with an Agilent RNA 6000 Nano Chip G2938‐80023.Reverse‐transcription was performed with the SuperScript III First‐Strand Synthesis SuperMix  and followed by an RNAse H treatment. Quantitative polymerase chain reaction (PCR) was performed with the SYBRGreen Jumpstart Taq Ready Mix (Sigma‐Aldrich) with 200 nM of the reverse and forward primer  was determined and the relative abundance was calculated according to the following formula:For each gene, the cycle threshold (relative_abundance=2(ctHKG−ctGOI)=2ctHKG2ctGOI where HKG = house‐keeping gene  and GOI = gene of interest .The samples were embedded in paraffin and 5 µm slices were cut with a rotary microtome. The standard staining was Safranin O 0.01% (w/v) with Fast Green 0.1% (v/v) as a counterstain. The immunohistochemical detection of type I and II collagens was achieved using a fully automated immunohistochemistry stainer . For type I collagen staining, antigen retrieval was performed with proteinase K 0.61 µg/ml (Leica) for 5 min at 37°C and the samples stained with a rabbit anti‐collagen I antibody at 0.7 µg/ml  followed by the Bond Polymer Refine Detection kit (Leica). For type II collagen staining, antigen retrieval was performed with chondroitinase ABC 0.5 U/ml (Sigma‐Aldrich) for 15 min at 37°C and the samples stained with a rabbit anti‐collagen II antibody  at 2.5 µg/ml followed by the EnVision + System‐HRP detection kit (Agilent).Statistical analysis was conducted using Graphpad Prism v7.00 . One‐way analysis of variance was performed with a Dunnet test to correct for multiple comparisons.The cells were treated with 0.3–1,000 ng/ml of IGF1, IGF2, BMP7, or sprifermin and 0.003–10 µM of CNP for 7 days; and the cell number, GAG production, and gene expression were measured. The cell number  and col2a1 , and an increase in acan expression . Although the effects of IGF1 on gene expression were variable across doses, col1a2 and col2a1 and acan expression increased compared with the control . Similarly, treatment with IGF2 also increased the expression of col1a2 and col2a1 and acan . BMP7 stimulated a significant increase in col1a2 (by 1.3‐fold at 100 ng/ml) and col2a1 (by twofold at 30 ng/ml) expression, but not at concentrations above 100 ng/ml. In contrast, the highest concentration of BMP7  stimulated a 2.6‐fold increase of acan expression. As with the cell number and GAG production, CNP treatment had little to no impact on the expression of cartilage‐related genes.The expression levels of the cartilage‐related genes To further compare the anabolic functions of IGF1, IGF2, BMP7, and CNP with that of sprifermin, bovine chondrocytes were cultured in a scaffold‐free 3D construct. For IGF1, IGF2, and BMP7, 300 ng/ml was used and for sprifermin, 100 ng/ml. This corresponded to the concentrations that elicited a substantial effect on cell proliferation and GAG release in monolayer. For CNP, 10 nM was chosen based on the previous results from others.In both experiments Fig. B and C, acan (by 3‐ and 12‐fold) and col2a1 (by 7.1‐ and 8.3‐fold) compared with the control. Acan expression was also observed to increase significantly with permanent exposure to sprifermin (by fivefold) in the second experiment while col2a1 expression was unaffected by the permanent exposure to sprifermin. Permanent exposure to sprifermin decreased col1a2 expression compared with the control (by 178‐fold in the first experiment and sevenfold in the second experiment). As a result, sprifermin increased the ratio of col2a1:col1a2 compared with the control with both cyclic  and permanent exposure (by 8.8‐ or 16.5‐fold). Cyclic exposure to IGF1 and IGF2 had no effect on the expression of acan, col2a1, or col1a2. However, with permanent exposure IGF1 significantly increased acan (by 2.7‐fold), col2a1 (by 7.3‐fold), and col1a2 (by 7.4‐fold) expression. IGF2 showed a similar profile although the differences compared with the control were not statistically significant. Cyclic exposure to BMP7 significantly increased the expression of col2a1 (6.1‐fold) and col1a2 (by 4.6‐fold) while permanent exposure significantly increased acan (by 17.6‐fold) and col2a1 (by 11.1‐fold) expression compared with the control. Permanent exposure to BMP7 also showed an increase in the ratio of col2a1:col1a2 (by 7.5‐fold), similar to that observed with sprifermin. CNP had very few effects on gene expression; only col2a1 expression was increased (by 5.6‐fold with cyclic exposure).Analysis of cartilage gene expression from the 3D constructs is shown in Figure mmp13, runx2, and col10a1, was evaluated  after permanent exposure to sprifermin in the first experiment while mmp13 expression was significantly reduced with both permanent (by 5.4‐fold) and cyclic (by 249‐fold) exposure in the second experiment. IGF1 and 2 both decreased significantly mmp13 expression with permanent exposure  and had no impact on runx2 expression. IGF1 but not IGF2 significantly increased col10a1 expression (by 7.4‐fold) with permanent exposure. In contrast to IGFs and sprifermin, BMP7 significantly increased runx2 expression with both cyclic and permanent exposure . BMP7 also significantly increased col10a1 expression (by 24‐fold) with permanent exposure and significantly reduced mmp13 expression with both the cyclic and permanent exposures . CNP did not alter mmp13 or col10a1 expression but cyclic exposure significantly increased runx2 (by 2.2‐fold).The expression of three hypertrophy markers, ted Fig. . Sprifercol10a1 expression observed with permanent exposure to BMP7 or IGF1. It is possible that the gene expression was not high enough to translate into detectable type X collagen deposition.Safranin O staining and staining for type I and II collagen of the 3D constructs was performed Fig. . The untThe treatment of osteoarthritic joints with an anabolic compound could drive regeneration and regrowth of the cartilage tissue, which is usually degraded as the disease progresses. The aim of this study was to compare the anabolic properties of several compounds reported to have positive effects on cartilage tissue—IGF1, IGF2, BMP7, and CNP—with those of sprifermin, a human recombinant FGF18, which is in clinical development for knee OA.col2a1 and acan expression. This is in accordance with the previously reported results.Col1a2 expression was also enhanced by these three growth factors. Sprifermin had a different profile; it induced a strong reduction of col1a2 expression by 59‐fold at the highest dose and a more moderate reduction of col2a1 expression by 7.6‐fold. GAG production was significantly reduced. Sprifermin was also the molecule that promoted cell proliferation the most, indicating as proposed before, that a strong proliferation might occur at the expense of matrix production.The anabolic compounds were first tested on bovine chondrocytes in monolayer and all of them except CNP increased cell proliferation. Furthermore, IGF1, IGF2, and BMP7 stimulated GAG accumulation in the culture medium as well as For the subsequent evaluation in the 3D culture, IGF1, IGF2, and BMP7 were used at 300 ng/ml, a concentration that elicited a substantial effect on proliferation and GAG accumulation in monolayer. Sprifermin was used at 100 ng/ml, a concentration that led to a strong proliferation and demonstrated a significant anabolic effect in 3D culture in previous studies.col2a1:col1a2 was increased with both exposures, while the expression of hypertrophy markers was unaffected, indicating that sprifermin favored the chondrocyte phenotype. These results confirmed similar findings obtained with porcine and human OA chondrocytes in the same culture model.In 3D culture, both permanent and cyclic exposure to sprifermin induced an anabolic response but the response was stronger with cyclic exposure. In addition, the ratio of col1a2 as previously reported,col2a1:col1a2 ratio. Regarding the hypertrophy markers, both IGFs decreased mmp13 expression as already described,col10a1 expression with permanent exposure.IGF1 and IGF2 both displayed a similar profile in 3D culture; cyclic exposure had no impact, but the permanent exposure resulted in an increased ECM production. The anabolic effect observed with permanent exposure is in accordance with the previous studies.acan and col2a1 but having no effect on col1a2. Interestingly, the cyclic exposure to BMP7 did elicit an increase of the expression of col2a1 but not acan, and in this case, col1a2 was elevated. It was also previously reported that BMP7 has no hypertrophic effect in bovine articular chondrocytes and may even have a hypertrophy‐suppressive effect in ATDC5 and human bone marrow stem cells of BMP7.Similar to other reports,Finally, CNP had almost no effect in 3D culture similar to that observed in the monolayer culture. In contrast to our findings, Waldman et al.,col1a2 expression both in monolayer and in 3D with permanent exposure. On the contrary, both IGFs and BMP7 increased col1a2 expression in monolayer and in 3D and displayed a stronger type I collagen staining compared with sprifermin. Furthermore, sprifermin did not increase the expression of any of the hypertrophy markers.This head‐to‐head comparison of sprifermin, IGF1, IGF2, and BMP7 enables direct comparison of the effects of these growth factors on the phenotype and anabolism of primary articular chondrocytes to be made. To our knowledge, it is also the first study where IGF1, IGF2, and BMP7 have been evaluated with a cyclic exposure compared with a permanent exposure. When comparing the four growth factors in terms of the chondrocyte phenotype, it appeared that sprifermin was the only one that decreased In terms of their anabolic effect, the four growth factors were equivalently effective: sprifermin with cyclic exposure, IGFs with permanent exposure and BMP7 with both exposures. However, showing efficacy with a cyclic exposure is of great advantage for potential OA therapy.OA is a localized disease and many therapeutic approaches use the local application, namely intra‐articular injection.This study presents some limitations. For instance, it is unclear how results obtained with bovine chondrocytes translate to the human situation; however, testing many conditions with human chondrocytes would have been challenging due to the low availability of this material. Another limitation is that only one independent experiment was realized for each culture condition and compound. However, we believe that testing the same molecules in different settings  enabled us to robustly compare the tested molecules.In conclusion, sprifermin and BMP7, but not IGF1 and IGF2, exerted an anabolic effect with cyclic exposure on bovine chondrocytes. This characteristic makes them suitable for intra‐articular treatment for OA without the need for a slow‐release formulation. In addition, sprifermin was the only tested compound that favored the chondrocyte phenotype by decreasing type I collagen and having no hypertrophic effect. Consequently, sprifermin is more likely than BMP7, IGF1, or IGF2 to result in the production of hyaline cartilage. The results of this study confirm that sprifermin presents a promising therapeutic option as an intra‐articular DMOAD.S.M. designed the experiments, acquired and analyzed the data, and drafted the paper. A.G. designed the experiments, analyzed the data, and critically reviewed the paper. S.L. participated in data acquisition and critically reviewed the paper. All authors have read and approved the final submitted manuscript.


In this study, these limitations are overcome by constructing a triphasic photocatalytic system, in which polymeric carbon nitride (CN) is immobilized onto a hydrophobic substrate, and the photocatalytic reduction reaction occurs at a gas–liquid–solid  triple interface. CN anchored onto the surface of a hydrophobic substrate exhibits an approximately 7.2‐fold enhancement in total CO2 conversion, with a rate of 415.50 μmol m−2 h−1 under simulated solar light irradiation. This value corresponds to an overall photosynthetic efficiency for full water–CO2 conversion of 0.33 %, which is very close to biological systems. A remarkable enhancement of direct C2 hydrocarbon production and a high CO2 conversion selectivity of 97.7 % are observed. Going from water oxidation to phosphate oxidation, the quantum yield is increased to 1.28 %.The activity and selectivity of simple photocatalysts for CO Access all areas: A triphase interfacial CO2 reduction system over a carbon nitride photocatalyst enables the reactant CO2 to reach the reaction interface directly from the ambient atmosphere, which simultaneously enhances the reaction rate and suppresses the competitive hydrogen evolution. To overcome kinetic limitations and suppress the hydrogen evolution reaction, numerous efforts have focused on the improvement of pristine photocatalysts, by methods such as loading cocatalysts,2 conversion process. Previous studies found that the availability of excess protons (H+) and low concentration of CO2 at the reaction interface lead to unsatisfactory activity and selectivity of the photocatalytic CO2 reduction system.2 photoreduction, the availability of CO2 at the reaction interface is dependent on its mass transfer through the water phase.2 molecules in water thereby strongly hinder the surface catalytic process of CO2 photoreduction.Ever‐increasing consumption of fossil fuels along with the massive emission of carbon dioxide CO has gene2 photoreduction, a simple and sustainable approach is developed by constructing a triphase (gas–liquid–solid) interfacial photocatalytic system. The photocatalysts are immobilized on the surface of a carbon fiber substrate , liquid (water), and solid . Such a reaction system then allows the continuous delivery of CO2 molecules from the gas phase to the reaction interface via its hydrophobic channels, instead of the slow diffusion through the liquid phase. As a result, the accessibility of CO2 molecules to the photocatalyst is greatly increased, which subsequently enhances the rate of the reaction between CO2 and photogenerated electrons, thereby diminishing electron–hole recombination and increasing charge utilization. Finally, the activity and selectivity of photocatalytic CO2 conversion is remarkably improved.In this study, to overcome the limitations of the conventional liquid–solid diphase system of COe Figure . The con2 is directed through the hydrophobic substrate up to the CN particles, resulting in the formation of a gas–liquid–solid triphase boundary zone. Such a framework then secures the supply of both abundant water and CO2 molecules to drive overall CO2 photoreduction. Field‐emission scanning electron microscopy (FESEM) allows direct observation of the interface between immobilized CN photocatalysts and carbon fiber substrates  nanosheets onto the surface of a carbon fiber (CF) fleece with porous structure and different wettability. The contact angles (CAs) of the superhydrophilic, hydrophilic, and hydrophobic CF substrates are approximately 0°, 37.5°, and 112°, respectively  patterns of CF, CN/CF1, CN/CF2, CN/CF3, and CN are shown in Figure l–S) triphase reaction interface was employed . As a result, a spectrum of product molecules is found , as well as that of CH4, over the hydrophobic substrate is significantly higher than that over the hydrophilic substrate. It can be concluded that CN immobilized onto the hydrophobic substrate experiences a continuous supply of CO2 molecules, thus making CO2 reduction processes much more effective than H2 generation under the given conditions of no metal cocatalyst. Moreover, increasing the loading amount of CN would further enhance the photocatalytic activity while maintain the high selectivity (see data of CN/CF4 in Table S1).The photocatalytic activity and selectivity of as‐prepared CN/CF photocatalysts were investigated under simulated sunlight irradiation in the absence of any sacrificial agent and co‐catalyst. The novel reaction environment of gas–liquid–solid  by CO2 photoreduction over CN/CF3 in the triphase system, which is higher than the stoichiometric yield calculated from the given product distribution (644.34 μmol m−2 h−1). This deviation is reasonable within experimental error and might be related to undetectable species, such as methanol and ethanol, which end up dissolved in the water phase.In addition, the production of O2 was employed, and the hydrocarbon products carrying the isotopes 13C, 12C, and 2H (D) were detected by GC‐MS for the triphase system . Clearly, the signals m/z=16.1 (12CH4) and m/z=17.1 (13CH4) were dominant in the GC‐MS spectra for photocatalytic 12C‐labeled CO2 and 13C‐labeled CO2 reduction, respectively  were performed for photocatalytic CO2 reduction over CN/CF3 . The signals for molecular ethylene are at m/z=30.1 and m/z=34.1 when 13CO2 reacts with H2O and D2O, respectively. The signals at m/z=29.1 and m/z=32.1 are stronger those at m/z=30.1 and m/z=34.1, owing to the higher stability of the molecular ions  and a quantum yield of 1.28 % were achieved. In this case, the synthetic photosynthesis even outperformed biological photosynthesis, in spite of its simplicity. Perphosphates therefore represent a valuable route of investigation, but also can be thermally or catalytically decomposed to liberate dioxygen to terminate product formation.There are still a number of means to improve the activity that were not explored in these first‐generation experiments. We still consider the rate‐determining step to be the four‐electron oxidation of water to dioxygen, which here proceeds in the absence of a cocatalyst. A kinetically much simpler reaction than O2 reduction system based on the gas–liquid–solid reaction interface allows efficient and continuous delivery of CO2 molecules to the catalyst surface and inhibits the hydrogen evolution reaction. The photogenerated charge carriers are efficiently utilized, resulting in significantly enhanced activity and selectivity in the photocatalytic CO2 reduction. In particular, the CN anchored onto the surface of a hydrophobic substrate (CN/CF3) exhibits about 7.2‐fold enhancement in the total CO2 conversion with 415.50 μmol m−2 h−1 as compared to the CN anchored onto the surface of superhydrophilic substrate (CN/CF1). This is accompanied by a CO2 conversion selectivity of 97.7 %, the remainder being the otherwise dominant H2 evolution. This product yield for photocatalytic CO2 reduction is also clearly superior to that with the conventional diphase system, and could be further enhanced by simplifying the photooxidation process from four‐electron dioxygen generation to perphosphate formation, with a CO2 conversion of 1413.85 μmol m−2 h−1 and a quantum yield of 1.28 %. Interestingly, a higher CO2‐based reactant flux also improved C2 hydrocarbon production, which reflects the higher chance of intermediate C1 species recombining at the catalyst surface. This work provides a platform to explore further interfacial architectures in system engineering of highly active semiconductor photocatalysts.In summary, a triphase interfacial photocatalytic COThe 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.