In the experiment, the particles were initially coated with a few tens of nanometers of TiO2 with a strong shading effect, followed by a further coating of Ag of the same thickness with a similar shading performance. Not only did this achieve a lighter color, but there was no reduction in the magnetic properties of the material after the application of the coating. Scanning electron microscopy (SEM), scanning electron microscopy and energy-dispersive spectroscopy (SEM-EDS), X-ray diffractometry (XRD), and other methods were used to study the changes in morphology and composition before and after the magnetic material was coated. A magnetic tester was used to study the changes in magnetic strength before and after the magnetic material was coated.Magnetic materials are being increasingly used in anti-counterfeiting coatings, but the dark colors of magnetic materials greatly limit their applications. This necessitates the development of light-colored magnetic materials. In this study, the heterogeneous precipitation method was used to deposit a layer of titanium dioxide (TiO Magnetic materials are being increasingly used in anti-counterfeiting coatings, but the dark colors of magnetic materials greatly limit their applications. TiO2 is a photocatalyst that can be used in solar cells, water splitting, pollutant degradation, and other applications.5,6 It is used in photocatalysis,7,8 self-cleaning coatings,9 and hydrogen production.10,11 Photocatalytic sensors12,13 have a wide range of potential applications. Additionally, TiO2 has excellent opacity, whiteness, and brightness and is considered to be the best white pigment currently available; it has good hiding power and strong adhesion, and is an exceptionally white inorganic pigment used in paints14,15 and as a matting agent in enamels. Metallic silver (Ag), which is silvery-white, not only has good electrical and thermal conductivity, but also excellent shading properties.16–18 The color of magnetic materials, whether these are metal oxides or metal materials, is relatively dark, which greatly limits their applications. The use of light-shielding materials to mask the color of magnetic materials has therefore become a topic of great interest in recent years. Since the shortest wavelength of visible light is approximately 400 nm, it is necessary to coat the surface of the magnetic material with a layer of high-light-shielding material with a thickness of more than 400 nm in order to conceal the color of dark magnetic materials. TiO2 coating on the surface of magnetic materials can provide a good light-shielding effect, but requires a very thick coat of TiO2, which greatly reduces the magnetic properties of magnetic materials. Furthermore, TiO2 has a strong effect on organic matter. Photodegradation can also be problematic;19 if a magnetic material coated with TiO2 is directly applied to an organic polymer, the organic polymer in contact with the magnetic material will be degraded.20,21 This study examines the use of an initial coating of a layer of tens of nanometers of TiO2 on the surface of the magnetic material, followed by a layer of tens of nanometers of Ag. Not only does this avoid the degradation of organic matter by TiO2, but it also makes the magnetic material well. Thus, the color becomes lighter without affecting the magnetic properties.Composite particles with a core–shell structure have important application prospects in catalysis, magnetism, and composite materials science.2.2.13; analytical grade, Aladdin reagent); ammonia water  Chemical Reagent Co., Ltd); and absolute ethanol  were used.Iron–cobalt magnetic material (Fe–Co50); tetrabutyl titanate ; ethyl orthosilicate ; polyvinylpyrrolidone ; trimethylhexadecylammonium bromide ; silver nitrate (AgNO2.2A scanning electron microscope (SEM) ; an energy-dispersive spectroscopy (EDS) annex for the scanning electron microscope ; an X-ray diffractometer ; and a 7400 series vibrating sample magnetometer  were employed.2.32.3.12).A total of 3 g of Fe–Co50 was dispersed in a solution of 50 mL ethanol and 8 mL deionized water. Following the addition of 2 mL 1% CTAB, the above solution was transferred to a 250 mL three-necked flask, and stirred for 20 min in an ice-water bath. Then, 3 mL TBT was placed in a constant-pressure dropping funnel, which slowly it into the Fe–Co50 solution. After the addition was complete, the solution was stirred for another 2 h in an ice-water bath. Subsequently, the temperature of the reaction mixture was raised to 50 °C, and the solution was allowed to stir and react for 2 h. Then, the temperature was raised to 75 °C and the solution was stirred again for 4 h, at which point the reaction was stopped. The reactants were ultrasonically cleaned with ethanol 3–5 times and dried in a blast drying box at 80 °C to obtain a titanium-dioxide-coated magnetic material . 2@Ag.A total of 10 g of Fe–Co50@TiO3.3.12, which is deposited on the surface of Fe–Co50 magnetic particles. The hydrolysis reaction rate of TBT is known to have a positive correlation with temperature. The ice water bath conditions can decrease the hydrolysis rate and regulate TiO2 production. However, the hydrolysis reaction of TBT is relatively fast and non-selective. If it is not induced and controlled, TiO2 particles will be produced in the solution, and the Fe–Co50 surface will not be coated. CTAB forms micelles in the ethanol–water system and binds water to the micelles of the tert-butyl hydrophilic group. The surface of the Fe–Co50 magnetic particles is a polar surface. After CTAB is added, it forms micelles on, and binds water to, the surface of Fe–Co50, causing the hydrolysis reaction of TBT to proceed only on the surface of Fe–Co50. This achieves better control of the reaction in a heterogeneous phase.TBT undergoes a hydrolysis reaction in the ethanol–water system to generate TiO2 on Fe–Co50  was used to quantitatively study the coating of TiOCo50 see . The worared see . The ato2 under different TiO2 coating amounts. For 3 g Fe–Co50, when the dosage of TBT is gradually increased from 1 mL to 6 mL, the TiO2 coating amount gradually increases, the coating thickness gradually increases, and the color gradually becomes lighter. Increasing the coating amount of TiO2 is a gradual process resulting in a change in the apparent color of Fe–Co50, but the color of Fe–Co50 will not fundamentally change by increasing the TiO2 coating amount alone. The amount of TBT compared to Fe–Co50 added  is approximately VTBT : WFe–Co50 . Under the above feed ratio, the coating thickness is in the range of 20–30 nm (confirmed by SEM as discussed in Section 3.3). Above a TBT dosage of VTBT : WFe–Co50 = 1 : 1 (mL : g), the color of Fe–Co50@TiO2 hardly changes when the coating amount is increased. This has been used as the feed ratio from this point on, unless specified otherwise.Samples of the magnetic materials with different coating thicknesses were transferred to vials and photographed. 3.22; the role of PVP is to control the growth direction and crystal size of the silver crystals to form a dense coating structure.The silver ammonia solution of AgNO3 reacts with glucose to produce elemental silver (Ag). The addition of PVP during the reaction can induce Ag deposition on the surface of Fe–Co50@TiO2 coating, increasing the coating amount of TiO2 has little effect on the color change. Therefore, Fe–Co50@TiO2 was prepared with a feeding ratio of VTBT : WFe–Co50  as the precursor material for AgNO3 coating. As for the TiO2 layer, the elemental composition of the Fe–Co50@TiO2@Ag magnetic material following the AgNO3 coating of Fe–Co50@TiO2 was detected by SEM-EDS. Using 10 g samples of Fe–Co50@TiO2, the coating conditions of different AgNO3 additions were studied. The ordinate of vs. Fe , and the abscissa is the feed mass of AgNO3 . The molar ratio of Ag to Fe is used to measure the coating thickness of Ag on Fe–Co50@TiO2. It can be seen from vs. Fe increases with the addition of AgNO3. The coating amount of Ag on the surface of Fe–Co50@TiO2 shows almost a linear ratio with an increase in AgNO3 feeding amount.From the results discussed in Section 3.1, it is known that above a certain thickness of TiO2@Ag magnetic materials prepared with different amounts of AgNO3 were put into vials, and the color changes for different coating amounts are shown in 2, with coating masses of AgNO3 of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 g, respectively. It can be seen from 3vs. Fe–Co50@TiO2 reaches W3AgNO : W2Fe–Co50@TiO = 0.7 : 10 (g : g), a limit is reached and despite the feed of AgNO3 being increased, the color of the magnetic material does not change significantly. At this point, the color of the Fe–Co50@TiO2@Ag magnetic material is already white or light gray. As AgNO3 is relatively expensive, the most cost-effective approach, which still ensures that the color of the magnetic material is light, is using a AgNO3 : Fe–Co50@TiO2 ratio of 0.8 : 10 (g : g), such as in Fe–Co50@TiO2 coating amount, but after reaching a limiting coating amount, the color change is not obvious. When the coating thickness of Ag reaches a certain limit, the change in color of Fe–Co50@TiO2@Ag magnetic material also plateaus. In order to save costs and avoid reducing the magnetic properties of these materials, this study aims to determine the optimal coating amount of TiO2. In this experiment, when the feeding ratio of AgNO3vs. Fe–Co50@TiO2 was selected as W3AgNO : W2Fe–Co50@TiO = 0.8 : 10 (g : g), the color comparison chart of Fe–Co50@TiO2@Ag under different TiO2 coating thicknesses was studied , such as in 2 coating no longer has a significant color change. It will only result in an increase in production cost and a reduction in the magnetic properties of the material.The previous experiment showed that the color of Fe–Co50 magnetic material starts to change with increasing TiOdied see . Fig. 6; the SEM image shows that the TiO2 coating on the surface of Fe–Co50@TiO2 is complete, dense, and relatively uniform. From the cracks in the figure, it can be seen that the coating thickness of TiO2 is in the range of 20–30 nm. 2@Ag. The feed ratio of AgNO3vs. Fe–Co50@TiO2 is W3AgNO : W2Fe–Co50@TiO = 0.8 : 10 (g : g). It can be seen from the Fe–Co50@TiO2@Ag SEM image that the Ag particles are densely coated on the surface of Fe–Co50@TiO2 with a small particle size. The particles are tightly and completely coated on the surface of the magnetic particles. The thickness is also in the range of 20–30 nm.SEM can directly observe the surface structure and morphology of a given material, and was used to study the surface coating of the magnetic material. The surface morphologies of Fe–Co50, Fe–Co50@TiO3.42 magnetic material under the condition of VTBT : WFe–Co50 = 1 : 1 (mL : g). It can be seen from the SEM-EDS mapping of Fe–Co50@TiO2 that TiO2 coats the Fe–Co50 surface uniformly.SEM-EDS can be used to perform qualitative and semi-quantitative analysis of the elemental composition of a material, and the distribution of the elements can be determined through surface distribution mapping. 2 @Ag magnetic material prepared under a feed ratio of silver nitrate vs. Fe–Co50@TiO2 of W3AgNO : W2Fe–Co50@TiO = 0.8 : 10 (g : g). The SEM-EDS surface distribution mapping clearly shows that Ag uniformly coats the surface of Fe–Co50@TiO2.3.52, and Fe–Co50@TiO2@Ag, respectively. There is no diffraction peak in the Fe–Co50@TiO2 X-ray diffraction curve, indicating that the particle size of TiO2 is very small, maybe less than 2 nm, and no larger particles are formed during the process of coating Fe–Co50 with TiO2. This means that the Fe–Co50 surface can be evenly coated with TiO2 particles. The red X-ray diffraction curve of Fe–Co50@TiO2@Ag shows obvious metal Ag diffraction peaks, indicating that the Ag particles are relatively large. From C1 and C2 in 3.6VTBT : WFe–Co50 = 1 : 1 (mL : g) for the preparation of the Fe–Co50@TiO2 precursor. Further, the feed ratio of AgNO3vs. Fe–Co50@TiO2, W3AgNO : W2Fe–Co50@TiO, was 0.4 : 10, 0.5 : 10, 0.6 : 10, 0.7 : 10, 0.8 : 10, 0.9 : 10, and 1 : 10 (g : g). Hc) and the saturation value of magnetization (Ms) of the Fe–Co50@TiO2@Ag materials with different coating thicknesses by using a 7400 series vibrating sample magnetometer. With increasing coating thickness, the magnetic performance decreased slightly, but due to the reasonable control over the coating thickness in the experimental design, the performance of the magnetic material did not decrease significantly.Magnetic materials are remarkable functional materials. The coercivity and remanence of these materials are the basis for their wide application. Therefore, changing the color of a magnetic material must not affect its own magnetic properties. In this study, after making the color of the magnetic material lighter by surface coating, the influence of different coating thicknesses on the weakening of magnetic properties is studied. The feed ratio of TBT to Fe–Co50 was 4.2 with excellent light-shielding qualities, followed by a layer Ag of the same excellent light-shielding ability. The Ag particles formed a core–shell structure with complete surface coating. Upon appropriately adjusting the coating thickness, the color of the coated magnetic particles became lighter without the magnetic properties of the coated magnetic particles significantly deteriorating. By adjusting the feed ratio, it was found that the optimal feed ratio of TBT vs. Fe–Co50 was 1 : 1 (mL : g), and the optimal feed ratio of AgNO3vs. Fe–Co50@TiO2 was 0.8 : 10 (g : g). After the Fe–Co50 magnetic material was coated with TiO2 and Ag, the element surface distribution was characterized by SEM-EDS, which showed that the magnetic particles were perfectly coated by TiO2 and Ag. The magnetic properties of Fe–Co50@TiO2@Ag with different Ag coating thicknesses were tested, and it was found that the magnetic properties of the material did not significantly decrease when a lighter color was achieved.The surface of magnetic spherical particles was coated by an improved heterogeneous precipitation method. The surface of the magnetic spherical particles was first coated with a layer of TiO2 has the best opacity, whiteness, and brightness; thus, coating the surface of magnetic materials with TiO2 can provide a good light-shielding effect, but the required amount of TiO2 coating results in a relatively thick layer. Furthermore, TiO2 can photodegrade organic matter. By coating the deposited layer of TiO2 with a second layer of Ag, which has no degrading effect on organic matter, we are able to not only reduce the degradation of organic matter by TiO2, but also make its color lighter. This work offers a solution to the difficult problem of covering the surface of magnetic particles completely, presents a method for preparing light-colored magnetic materials that broadens the application range of magnetic materials, and has high application value. Furthermore, it provides a new preparation method for the complete coating of the surface of a granular material and the formation of a core–shell structure with a complete surface.The shortest wavelength of visible light is approximately 400 nm. In order to conceal the surface color of dark magnetic materials, the light-colored coating must be of a thickness exceeding 400 nm. TiOThere are no conflicts to declare.


We hypothesized that rapamycin (Rapa), acarbose (ACA), which both increase mouse lifespan, and 17α‐estradiol, which increases lifespan in males (17aE2) all share common intracellular signaling pathways with long‐lived Snell dwarf, PAPPA‐KO, and Ghr−/− mice. The long‐lived mutant mice exhibit reduction in mTORC1 activity, declines in cap‐dependent mRNA translation, and increases in cap‐independent translation (CIT). Here, we report that Rapa and ACA prevent age‐related declines in CIT target proteins in both sexes, while 17aE2 has the same effect only in males, suggesting increases in CIT. mTORC1 activity showed the reciprocal pattern, with age‐related increases blocked by Rapa, ACA, and 17aE2 . METTL3, required for addition of 6‐methyl‐adenosine to mRNA and thus a trigger for CIT, also showed an age‐dependent increase blunted by Rapa, ACA, and 17aE2 . Diminution of mTORC1 activity and increases in CIT‐dependent proteins may represent a shared pathway for both long‐lived‐mutant mice and drug‐induced lifespan extension in mice. ACA, Rapamycin and 17alfa Estradiol downregulate mTORC1 activity and upregulation of the Cap‐Independent Translation leading to increases in the levels of cap‐Independent associated proteins , suggesting a possible mechanism for lifespan extension. The results /S6 ratio and, by implication, mTORC1 activity. ACA and Rapa (both early and late) reduce this ratio, and 17aE2 does so in male mice only /4EBP1 ratio showed the same pattern of age, sex, and drug effects: age‐related increases in both sexes, reversed or blunted by Rapa and ACA in both sexes and by 17aE2 in males only, regardless of the age at which drug exposure was initiated. In contrast to the phosphorylation ratio for S6, the age and drug effects for the 4EBP1(pT37)/4EBP1 ratio do not reflect changes in the phosphorylation level of 4EBP1(pT37) per se, but instead reflect changes in the level of total 4EBP1 protein  levels as noted previously by us and others in several mouse models /S6 and 4EBP1(pT37)/4EBP1 ratios, suggesting increases in mTORC1 activity  from age or drug treatments  and cellular stress resistance, such as MGMT and NDRG. The CIT‐dependent increases in translation of these mRNAs might be caused by one or several factors in combination, potentially including a decline in cap‐dependent translation and/or increases in the pathways involved in adding, reading, or removal of m6A residues in the 5′UTR of the corresponding mRNA /S6 and the ratio of 4EBP1(pT37)/4EBP1  17aE2 can significantly block or reverse these mTORC1 changes, suggesting that inhibition of mTORC1 and cap‐dependent translation may contribute to their ability to maintain or restore the high levels of CIT seen in young mice. It is notable in Figures (pS235) while the 4EBP1 ratio reflects alterations, instead, in the total amount of 4EBP1 protein, rather than its phosphorylated form 4EBP1(pT37). We suspect that the use of mice fasted for 18 h. prior to euthanasia diminishes the phosphorylation level of 4EBP1 levels (Dominick et al., We interpret the age‐ and drug‐related changes in phosphorylation ratio for S6 and 4EBP1 as evidence for effects on mTORC1 function, but we note that the S6 ratio reflects alterations in S6p35 while Several slow‐aging mouse models, at 6 months of age, show age‐specific upregulation of the m6A pathway responsible for the methylation in position 6 of specific adenosine residues in mRNAs and upregulation of CIT (Ozkurede et al., 44.1Genetically heterogeneous UM‐HET3 mice were produced by a four‐way cross between CByB6F1 mothers and C3D2F1 fathers and housed as previously described (Garratt et al., 4.2Tissues were harvested at the age indicated in Figure 4.3t tests to evaluate specific contrasts of interest, that is, Young vs Old controls, and Old controls vs each drug‐treated group. Significance was evaluated using a criterion of p = 0.05 without adjustment for multiple comparisons.All statistics were carried out in GraphPad Prism version 7.0. Data from animals treated with ACA, Rapa, and 17aE2 were analyzed separately, but the same young and aged control animals were used in each analysis. To normalize between different Western blots, we use young females as a reference as we previously described (Ozkurede et al., The authors have declared that no competing interests exist.Ziqian Shen, Abby Hinson, and Gonzalo G. Garcia carried out the experiments and data analysis with the help of Richard A. Miller. Richard A. Miller and Gonzalo G. Garcia wrote the manuscript, while Richard A Miller helped to supervise the project.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Table S1‐S5Click here for additional data file.


The azetidine moiety is a privileged motif in medicinal chemistry and new methods that access them efficiently are highly sought after. Towards this goal, we have found that azabicyclo[1.1.0]butyl carbinols, readily obtained from the highly strained azabicyclo[1.1.0]butane (ABB), can undergo divergent strain‐release reactions upon N‐activation. Treatment with trifluoroacetic anhydride or triflic anhydride triggered a semipinacol rearrangement to give keto 1,3,3‐substituted azetidines. More than 20 examples were explored, enabling us to evaluate selectivity and the migratory aptitude of different groups. Alternatively, treatment of the same alcohols with benzyl chloroformate in the presence of NaI led to iodohydrin intermediates which gave spiroepoxy azetidines upon treatment with base. The electronic nature of the activating agent dictates which pathway operates. Azabicyclo[1.1.0]butyl carbinols can be converted into a diverse range of 1,3,3‐substituted azetidines or spirocyclic epoxides through either a semipinacol rearrangement or via iodohydrin formation followed by spirocyclization. The reaction pathway that operates is determined by the electronic nature of the activating agent. The past decade has seen a surge in the application of azetidines in pharmaceutical drug candidates.1).butane .5, 6 Fm Scheme .[8a] A2 as a synthon for 1,3,3‐substituted azetidines by exploring alternative electrophiles which could also trigger strain‐release reactivity and considered the use of ketones and aldehydes (Scheme 5), subsequent N‐activation by a suitable electrophile could trigger a pinacol‐type rearrangement, cleaving the bridging C−N bond and releasing ring strain. A5 a resulted in the formation of chlorohydrin 10 in 83 % yield  to d Scheme . This red Scheme .5 a can be rationalised by the extent of positive charge build‐up at the electrophilic bridgehead carbon. When TFAA is used as an activator, the trifluoroacetyl group, being more electron‐withdrawing than the Cbz group, results in a greater build‐up of positive charge, thereby favouring the semipinacol reaction pathway. We postulated that the different counterions could also play a role in determining the outcome of the reaction, therefore, we investigated whether product formation was determined by the nucleophilicity of the counterion or by the nature of the activating group. This was achieved by performing the reactions in the presence of NaI in order to keep the counterion (iodide) constant , the semipinacol rearrangement dominated to give sulfonamide 7 a in 50 % yield with no nucleophilic addition observed. Thus, the outcome of the reaction is predominantly determined by the electronic nature of the activating group on nitrogen: the more electron‐withdrawing it is the more the semipinacol pathway is favored.The divergent reactivity of t Scheme .[16] O2O were improved by switching the solvent to CH2Cl2 and performing the reaction at −78 °C . In the case of Tf2O, the yield was further increased by the addition of 2,6‐lutidine. We then explored the scope of the reaction using both sets of conditions (A and B) with a broad range of ABB‐carbinols  also worked well, giving compounds 6/7 d–f in good yields—again exclusive migration of the aryl group was observed. However, 4‐pyridyl carbinol 5 g was found to be a poor substrate, as the reaction was unsuccessful with TFAA and low yielding with Tf2O.For non‐symmetrical alcohols bearing an aryl and methyl group, in all cases, and under both sets of conditions, we found exclusive migration of the aryl group to give azetidines 6 h–k and 7 h–k were obtained after ring expansions from ABB‐carbinols bearing 4‐, 5‐, 6‐, and 7‐membered rings in yields of 27–54 % using TFAA (method A) and 50–87 % using Tf2O (method B). The lower yields when using TFAA were attributed to competing nucleophilic addition of trifluoroacetate due to the lower migratory aptitude of alkyl groups.5 l gave azetidines 6/7 l with an intriguing 2,6‐diazaspiro[3.4]octane core in 48 % and 63 % yields, respectively.2O (method B), exclusive migration of the more substituted alkyl group6/7 m, the latter in almost quantitative yield.Cyclic ketones were also explored as the semipinacol rearrangement would result in a ring expansion to give valuable spirocyclic azetidine scaffolds. Azetidines 6/7 n in 51 % and 84 % yields, respectively.6/7 o,p in excellent yields. However, comparing Ph/Cy resulted in high selectivity but only in the case of TFAA to give 6 q; Tf2O gave a 1:1.5 ratio of products 7 q/7 q′, now in favour of Cy migration. In both cases, the yield of the ketone was low. In fact, the main component of the latter reaction was spirocyclic epoxide 13, formed in 66 %. Trifluoromethyl carbinol 5 r was tested and as expected,6 r was formed in only 25 % yield with competing nucleophilic addition of trifluoroacetate observed. Selectivity for the semipinacol rearrangement was much higher with Tf2O (conditions B), giving azetidine 7 r in 72 % yield.We subsequently compared the migration of a Ph group over other substituents, including more substituted alkyl groups and other functional groups. Comparing Ph with H, we found that the Ph group migrated exclusively to give aldehydes 6 s and 7 s were obtained from ABB‐carbinol 5 s derived from benzophenone in 42 % and 90 % yields under conditions A and B, respectively. In the case of 6 s, the lower yield observed was due to competing nucleophilic addition. Finally, comparing Ph/p‐MeOC6H4, we found that the more electron‐rich aryl group migrated preferentially,6 t/6 t′ and 1.3:1 ratio of 7 t/7 t′ were obtained under conditions A and B, respectively.Azetidines 2O, but the selectivity is lower since it induces a faster reaction. In the case of the especially hindered substrate 7 q, containing both phenyl and cyclohexyl migrating groups, no selectivity is observed. Here, C−C bond rotation of the hindered substrate is likely to have a higher barrier than 1,2‐migration,2O.The commonly observed relative migratory aptitude in semipinacol rearrangements17, 2013 from 5 q under conditions B was intriguing, and we were keen to establish whether this pathway could be promoted more generally. We reasoned that iodohydrin 11, formed from the reaction of 5 a with CbzCl and NaI . Due to its instability on silica gel (as with 8 d), purification of the intermediate iodohydrin was necessary in order to isolate analytically pure 8 p.We then extended this protocol to produce a range of spirocyclic epoxides from selected ABB‐carbinols Scheme . Employi5 r, CbzCl was found to be unreactive, but treatment with TFAA in the presence of NaI led to selective formation of the iodohydrin intermediate. After the addition of potassium carbonate, epoxide 8 r was isolated in 49 % yield. The high selectivity for iodohydrin formation from 5 r contrasts with the reactivity of 5 a, where treatment with TFAA in the presence of NaI resulted in the formation of both iodohydrin 12 and the semipinacol product 6 a. This is a result of the slower rate of the semipinacol rearrangement with the trifluoromethyl ABB‐carbinol 5 r. Despite the reaction of 5 a with TFAA/NaI leading to a mixture of 12 and 6 a, treating this mixture with potassium carbonate led to the formation of epoxide 14 in 38 % yield. Interestingly, other activators could also be employed with NaI. Tosyl chloride (TsCl) behaved similarly to CbzCl to give sulfonamide 15 in 87 % yield, whereas di‐tert‐butyl dicarbonate (Boc2O) was slower to react and required heating to activate ABB‐carbinol 5 a. Under these conditions with Boc2O, cyclization of the iodohydrin occurred without additional base to give epoxide 16 in 57 % yield.In the case of trifluoromethyl ABB‐carbinol 2O) induce a semipinacol rearrangement in ABB‐carbinols to give either amide or sulfonamide azetidines. The semipinacol rearrangement proceeds with migration of the group best able to stabilize the positive charge, so follows the order aryl > substituted alkyl > less substituted alkyl. Even electron‐deficient aromatics and heteroaromatics migrate in preference to a methyl group. When two alkyl groups are present, the semipinacol rearrangement is much slower and in the case of TFAA as the activator, nucleophilic addition of the counterion begins to compete, leading to lower yields. However, switching to Tf2O as the activator allows the semipinacol rearrangement to dominate, leading to good reaction yields.We have discovered novel, divergent reactivity of azabicyclo[1.1.0]butyl carbinols, which are themselves easily obtained from the reaction of azabicyclo[1.1.0]butyllithium with carbonyl compounds. We found that strongly electrophilic activating reagents  should be addressed to the authors.SupplementaryClick here for additional data file.


A finite chain of thirty‐one paramagnetic centers is reported, synthesized by reaction of hydrated chromium fluoride, copper carbonate and pivalic acid in the presence of 1,4,7,10‐tetrazacyclododecane (cyclen). Magnetic studies show predominantly anti‐ferromagnetic exchange leading to a high density of low‐lying spin states and large saturation field. The longest finite chain of metals is reported and the magnetic properties studied and modelled. The designed approach to the synthesis of polymetallic complexes often uses rigid ligands with donor atoms organised to bind to metal sites with specific geometries.5, 6 TAnother route recognises that there are always chemical factors that are outside our complete control, and allowing such factors to play a part in reaching new structures. In this paper we take a well‐studied system,n‐propylamine, we form in very high yield a compound [nPr2NH2][Cr7NiF8(O2CtBu)16].2F(O2CtBu)2} fragment, which can oligomerise readily. The eight‐metal ring is the smallest closed structure, and hence favoured by entropy. Entropic control matches the reaction temperature of 140° C in molten pivalic acid.We have been studying the formation of cyclic and acyclic complexes from hydrated chromium fluoride reacted with pivalic acid in the presence of an amine and a divalent metal ion that favours a regular octahedral coordination environment, typically nickel(II).xCu2} }2Cr12NiF20(O2CtBu)22] which we have described as a molecular seahorse, 1.II leads to it acting as a terminal group and preventing ring closure. Another factor is the crystal field stabilisation energy for nickel(II); once bound to the cyclen it tends to stay bound and will adopt a six‐coordinate geometry.Inclusion of copper(II) in place of nickel(II) leads to a series of larger rings {CrII as the divalent ion and cyclen as the template introduces a further degree of uncertainty in our design and here it leads to a remarkable result. The reaction of chromium(III) fluoride hydrate, cyclen, basic copper carbonate, and pivalic acid at 140–160 °C for 5 h produced [Cu(H2O)(cyclen)]2[Cr24Cu5{Cu(cyclen)}2F40(O2CtBu)50], 2. It was obtained as an isolated product with low but reproducible yield, and can be separated from the neutral byproduct [CrF(O2CtBu)2]8 by extraction or crystallization. Crystals can be grown from a mixture of Et2O/MeCN solvent. The structure of 2  that links to Cu2. There is then a {Cr3} (Cr6…Cr8) that links to Cu3, and finally a {Cr4} chain (Cr9…Cr12) that links to the central Cu4 site. We have only previously seen more than one Cr‐chain in the same molecule in a {Cr11Cu2} ring.The asymmetric unit comprises one half of the oligomer, with the central copper (Cu4) residing on an inversion centre. There are seven Cuz‐axis for the CuII site. Bond length ranges are given in Table Each Cr⋅⋅⋅Cr contact in the three distinct chains is bridged by a fluoride and two pivalates, the same motif we have seen in previous rings and chains.13, 15Cu2 is bridged by two fluorides and a pivalate to Cr5 and by a single fluoride and two pivalates to Cr6. Two of the fluorides are on the elongated Jahn–Teller (JT) axis. Cu3 is bridged to Cr8 by a single fluoride and single pivalate, and to Cr9 by a fluoride and two carboxylates; this latter fluoride is on the JT axis. Cu4 is on the inversion centre and is bridged to Cr12 by two fluorides and a single pivalate. The JT‐axis for Cu4 is to two F‐atoms.+ cation, which forms N‐H⋅⋅⋅F hydrogen bonds to the chain with N⋅⋅⋅F separations of 2.8–2.9 Å.Cu5 is part of a [CuF(cyclen)]mer‐arrangement of three O and three F donors, while Cu4 is six coordinate with a trans‐arrangement of four F and two O donors. This capability of CuII to adopt five different coordination environments in one complex is a major reason for the formation of 2. All the CrIII sites are six‐coordinate, with Cr2, Cr3, Cr3, Cr4, Cr6, Cr7, Cr9, Cr10 and Cr11 having a cis‐arrangement of two F and four O donors. Cr5, Cr8 and Cr12 have a mer‐arrangement of three O and three F donors. Cr1 is unique in having four F and two O‐donors with a cis‐geometry.The five unique copper sites have four distinct geometries. Cu1 and Cu5 are square pyramidal, the former bound to 4N and 1F and the latter to O4 and 1O. Cu3 is also square pyramidal but bound to two F and three O donors, as noted above the fluoride is on the apex of the square pyramid. Cu2 is six‐coordinate with a No significant intermolecular interactions were observed in the solid state, which is similar to related rings.1 and 2. In 1 all nickel sites are six‐coordinate and it is the coordinative flexibility of CuII that allows formation of the longer chain. Examining the core of 2 we can see an almost planar metal S‐chain {Cr8Cu3} running from Cu3 to its symmetry equivalent via two {Cr4} chains and Cu4  was measured from 300 to 2 K in a 5000 Oe field and the magnetisation (M) was measured from 0 to 7 T at various temperatures  methods, and the second enables quantum Monte Carlo (QMC) in order to evaluate thermal properties such as the magnetization as a function of temperature and applied field. For both types of calculations we employed the free ALPS library.[The Sea‐Serpent belongs to the largest spin systems of finite size; the Hilbert space dimension is 36,028,797,018,963,968 (without Cu5 and Cu5′). Exact diagonalization works up to 10 library.17, 18z‐axis on the Cu site, regardless of the number of carboxylate bridges. In Table 12Cu2} ring, where the exchange interactions were determined by three techniques: magnetometry, inelastic neutron scattering and tunnel diode oscillator measurements.3J and 5J were allowed to vary with g fixed at 2.0. The best fit was then achieved with J3=3 K and J5=10 K  is not justified.There are many assumptions here, perhaps chiefly that the exchange of Cu1‐Cr1 is equivalent to that of Cu2‐Cr6 or Cu3‐Cu9; this assumption is based on the exchange being dominated by the F‐bridge on the S=3/2. Of the many levels within 3 K of the ground state we targeted five for each magnetic quantum number . Firstly, the spectrum in frozen solution (1:1 CH2Cl2:toluene) and powder at 5 K are very similar. This suggests the structure of the chain and its supramolecular interaction with the cation containing Cu5 is maintained in solution. Secondly, there is copper hyperfine structure on the low field feature in the spectra. We believe this is due to the resonances from the isolated Cu5 complex. This lower field feature is centered at g=2.13 while the higher field feature is at g=1.960, 1.990, 1.990. The spectrum was simulated3} chain with g=1.960 and J=16 K and two CuII sites with g values of 2.075, 2.055, 2.145 and 2.065, 2.045, 2.135.The EPR spectra of 2 demonstrates the richness and complexity of the chemistry that is found when chromium fluoride, pivalic acid and a copper(II) salt are present.2 is low, but reproducible. There must be other oligomers formed that we have not crystallized to this point. It also suggests that inclusion of other N‐donor macrocycles may lead to still further unusual structures.The synthesis and structure of 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.


This paper describes the structure and catalytic properties of the VOx/ZrO2 catalyst. When using ZrO2 as the support, the catalyst shows six times higher turnover frequency (TOF) than using commercial γ-Al2O3. Combining H2-temperature programmed reduction, in situ Raman spectroscopy, X-ray photoelectron spectroscopy and theoretical studies, we find that the interaction between VOx and ZrO2 can facilitate the reduction of V–O bonds, including V PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, V–O–V and V–O–Zr. The promoting effect could be attributed to the formation of low coordinated V species in VOx/ZrO2 which is more active in C–H activation. Our work provides a new insight into understanding the structure–performance correlation in VOx-based catalysts for non-oxidative propane dehydrogenation.Supported vanadium oxide is a promising catalyst in propane dehydrogenation due to its competitive performance and low cost. Nevertheless, it remains a grand challenge to understand the structure–performance correlation due to the structural complexity of VO x species on ZrO2 with more reduced V–O bonds leads to improved catalytic activity for propane dehydrogenation.Low coordinated VO Due to the large-scale exploitation of shale gas, direct propane dehydrogenation becomes a particularly important method to produce propylene using propane as feedstock. Commercialized propane dehydrogenation plants generally utilize Pt-based and CrOx-based catalysts can be utilized in propane oxidative dehydrogenation (ODH).4–8 However, there is a trade-off effect between conversion and selectivity associated with over oxidation to COx, which prevents the ODH process from achieving high propylene yield. In contrast, better reactivity and selectivity, especially excellent regeneration stability can be achieved when supported VOx catalysts are used in the non-oxidative propane dehydrogenation (PDH) process.9–13 Nevertheless, the structure–performance correlation of VOx-based catalysts is still unclear due to the structural complexity of the supported VOx catalytic system. Previous studies have confirmed that several factors could influence the performance of VOx-based catalysts such as polymerization forms of VOx, chemical states of V and the identity of the support.14–18It is well established that VOx can be bonded to a support through the V–O–support interaction. The variety of supports could lead to significant changes in the catalytic properties of VOx. It is generally accepted that the support could affect reactivity by stabilizing the active sites and/or altering the electron state of active sites.19–22 The support effect has been extensively studied over VOx-based catalysts in the ODH process. Researchers have concluded that the lattice oxygen in VO and V–O–support (V–O–S) bonds is consumed in C–H activation and H2O is formed. Thus, support identities could affect the reaction by tuning the oxygen vacancy formation energy, which is confirmed by experiments and DFT calculations.23–25The support effect is an essential parameter because VO2 rather than H2O. Previous studies found that the reactivity of PDH is dependent on the bond strength of V–O–S.22 It is proposed that V–O–S bonds are active sites. In addition, the support identity also influences the behavior of carbon deposition on VOx based catalysts, which inversely affects activity and on-stream stability.12,18 Nevertheless, the effect of a support is still not clear because the structure of VOx and the VOx–support interface in a reduced state is still ambiguous.However, the support effect works in an entirely different way in the PDH process because not only active sites but also reaction mechanisms are distinct from those of the ODH process. V–O bonds directly catalyze C–H activation forming Hx-based catalysts for PDH and provide a new insight into understanding how support identity matters. We discovered that VOx/ZrO2, a well-known catalyst for ODH,26–28 has a much more superior PDH performance than commonly used VOx/Al2O3. The turnover frequency (TOF) is six times higher by loading VOx on ZrO2 than on Al2O3. The remarkable improvement of reactivity has not been reported in previous studies. Rate measurement of catalysts with gradient V loadings was employed to identify the active site of the VOx/ZrO2 catalyst. In situ Raman spectroscopic measurements, X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) calculations were used to determine the structure evolution of VOx under a reducing atmosphere, and they show that many more V–O bonds on ZrO2  are consumed during reduction. On this basis, we proposed that the facile reducing nature of V–O bonds promotes the formation of lower coordinated V species which accounts for C–H activation enhancement.Herein, we explore the support effect on the catalytic performance of VOx loaded on ZrO2 and Al2O3 are denoted as xVZr and xVAl where x represents the mass percentage of V, metal base). The bare ZrO2 support, 1VZr and 1VAl were characterized by X-ray diffraction (XRD). As shown in 2 is mainly composed of monoclinic phase  with small amount of a tetragonal phase , which is consistent with the previous report using the same preparation method.29 The same XRD pattern of 1VZr and pure ZrO2 indicates that VOx does not change the crystalline structure of ZrO2. In addition, for VOx loaded catalysts, only diffraction lines of the support could be detected  experiments were carried out over 1VZr, ZrO2 and 1VAl. The temperature where the C3H8 signal begins to drop is determined as the C–H activation temperature. As shown in 2 (481 °C) and 1VAl (491 °C). Moreover, the C–H activation temperature of 3VAl .35–37 In addition, Jeon et al. prepared V–Zr mix oxide and found that V incorporation into the ZrO2 bulk phase would promote generation of more Zrcus.38 Although in our VOx/ZrO2 system, VOx is supported on the surface of a support, not in the bulk phase, there still remains different possibilities for this superior performance of the VOx/ZrO2 catalyst: ZrO2 enhances the activity of VOx, VOx enhances the activity of ZrO2 (Zrcus), or a combination of the two. Therefore, it is essential to verify if the active phase is VOx or Zrcus in order to understand the origin of this dramatic performance improvement. To this end, various experiments were performed to figure out whether the catalytic performance is due to VOx or Zrcus or both of them.It has been proved that pure ZrOversus V loading is shown in x should be the active component. After this linearly increasing period, the C3H8 consumption rate does not change with increasing V loading because full VOx overlayers are formed at 2 wt% and crystalized V2O5 appears afterward as confirmed by the Raman spectra  profile if Zrcus is the active site.37,39,40 However, these phenomena cannot be observed in the VOx/ZrO2 system. No ZrO2 reduction peak can be observed up to 600 °C  is responsible for C–H activation. A potential method to solve this problem is Surface Organometallic Chemistry (SOMC),41–43 which provides access to producing well-defined isolated VOx only possessing VO and V–O–support. This direction still needs further investigation.The propane consumption rate  Fig. S1b. In addio 600 °C , while oe Fig. S5. These r2-TPR experiments were performed to test the reducibility and VOx–support interaction of the catalysts  of V. As listed in Table S3,x can be more readily and deeply reduced on ZrO2.Hatalysts . The mais Fig. S6. The red2 reduction for 30 min. V 2p core levels of 1VZr and 1VAl are displayed in 2/3 centers at 516.5 eV in 1VZr and 517.5 eV in 1VAl. This indicates that the valance state of V in 1VZr is lower than that in 1VAl. According to the deconvolution results of V 2p shown in 5+, V4+ and V3+, which is consistent with previous results.33,44,48 The fraction of V3+ is 48.6% in 1VZr, which is higher than 6.6% in 1VAl.To further identify the valance state of V, XPS investigation was carried out over 1VZr and 1VAl after H3+ is more active in the PDH reaction.14,33 However, seldom detailed analyzes have been conducted to identify the structure of VOx in a reduced state and the structure–performance correlation is not clear. Therefore, in situ UV-Raman spectroscopy, which is more sensitive to the surface VOx structure,49 was performed to monitor the evolution of VO, V–O–V, and the V–O–support.It has been elucidated in the former articles that VO in 1VZr during H2 reduction is shown in −1 is ascribed to V–O–V and V–O–Zr bonds.32,50 The band at around 1020 cm−1 is assigned to VO in dispersed VOx.27,32,51 The number of V–O–V and V–O–Zr bonds decreases substantially which implies that plenty of these bonds are consumed during reduction. In addition, nearly 60% of VO in 1VZr disappeared after reduction for 30 min. The comparative experiment of 1VAl is displayed in 2O3 support are located at 400–600 cm−1 and 910 cm−1 respectively.49 A sharp band at 1018 cm−1 is ascribed to VO.49 In contrast with V–O–Zr and V–O–V in the 1VZr sample, the V–O–Al band only decreases to 60% of that for fresh 1VZr and no change of the V–O–V band is detected which implies V–O–V bonds on the Al2O3 support cannot be reduced under H2 treatment. Meanwhile, 40% of VO in 1VAl is reduced, which is less than that in 1VZr. The result of in situ Raman spectroscopy confirms that not only VO and V–O–Zr, but also V–O–V can be deeply reduced in 1VZr which results in the deeper reduction degree of 1VZr observed in H2-TPR and XPS studies.The evolution of V–O–V, V–O–Zr and Vx and the support. It has been shown that a lower support cation electronegativity can result in a higher electron density of the V–O–S bond, which lead to these bonds being readily reduced.52,53 Along with the lower reduction temperature of VZr shown in the H2-TPR profile, it can be deduced that the interaction between VOx and ZrO2 weakens the V–O bonds thus resulting in the facile reduction nature of the VZr catalyst.The facile reduction nature of V–O bonds in the VZr catalyst can be interpreted by the lower electronegativity of Zr compared with Al, which causes the difference of the interaction between VO3+, Zn2+, Zr4+, etc.) with a low coordination number are more active for the reaction.54 As more V–O bonds are reduced in the VZr catalyst, we could deduce that lower coordinated V sites are more active for the C–H activation. Our recent DFT calculations also proved that coordinatively unsaturated VOx was more active in C–H activation55 while this work gives experimental evidence.Note that when O atoms are removed during the reduction period, the chemical environment of V changes, which leads to the formation of coordinatively unsaturated V. It is established that metal cations (Ga2(1̄11) and γ-Al2O3(100) surface as representatives36,55  and H2(g) as the reference, in these vanadium oxide dimers to confirm the different reducibilities they showed in our experiment results. As listed in O, V–O–V and the V–O–support. Four of five V–O bonds in VZr  have relatively low oxygen formation energies while only two of five in VZr do . The calculated oxygen vacancy formation energies are in good agreement with the Raman spectroscopy result that VO, V–O–V and V–O–Zr bonds in VZr can be reduced under a H2 atmosphere while only VO and part of V–O–Al are reduced in VAl.We calculated oxygen vacancy formation energy of different oxygen atoms, with the energy of H2O2/m-ZrO2(1̄11) and V2O3/γ-Al2O3(100) models to represent the catalyst structure in a reduced state  and two (one VO and one V–O–Al) V–O bonds with the lowest oxygen vacancy formation energies were removed from the initial dimeric V2O5 structure respectively. The propane dehydrogenation barriers over partially reduced VZr and VAl were computed and transition state (TS) geometries are shown in 3H8-TPSR tests.Since VZr and VAl have different degrees of reduction, we constructed Ved state . In termx can be readily reduced on ZrO2 because the interaction between VOx and ZrO2 facilitates the reduction of VO, V–O–V and V–O–Zr bonds. However, for the VAl catalyst, only VO and some V–O–Al bonds can be reduced. Thus, more low coordinated V species form in the VZr catalyst during reduction, and these low coordinated V species exhibit a better performance in PDH.Based on the experimental and theoretical results, we propose a structure–performance correlation of VZr and VAl catalysts, as shown in x loaded on ZrO2 through a simple incipient wetness impregnation method, which exhibits a dramatically improved performance compared with VOx loaded on Al2O3 for propane dehydrogenation. The TOF3H8C of 1VZr is determined to be 0.0161 s−1, which is almost six times higher than that of 1VAl.In summary, we prepared VOx species rather than it over ZrO2. Besides, combining in situ Raman and XPS spectroscopy results, we propose the enhanced C–H activation on VZr results from the facile reduction of VO, V–O–V and V–O–Zr bonds, thus producing deeply reduced and lower coordinated V species. DFT calculations also confirm that the C–H rupture energy barrier is lower for partially reduced VZr with low coordinated V species. Considering the dramatic performance achieved through the interaction between VOx and ZrO2, our work provides a new insight into high-performance VOx-based catalysts for propane dehydrogenation.We further prove that the remarkable reactivity of the 1VZr catalyst was attributed to the promotion of C–H activation over VOThere are no conflicts to declare.SC-011-D0SC00690D-s001


The circulating concentration of 1,25(OH)2D is a positive regulator of FGF23 secretion in bone, forming a feedback loop between kidney and bone. The importance of FGF23 as regulator of vitamin D metabolism is underscored by the fact that in the absence of FGF23 signaling, the tight control of renal 1α‐hydroxylase fails, resulting in overproduction of 1,25(OH)2D in mice and men. During recent years, big strides have been made toward a more complete understanding of the mechanisms underlying the FGF23‐mediated regulation of vitamin D metabolism, especially at the genomic level. However, there are still major gaps in our knowledge that need to be filled by future research. Importantly, the intracellular signaling cascades downstream of FGF receptors regulating transcription of 1α‐hydroxylase and 24‐hydroxylase in proximal renal tubules still remain unresolved. The purpose of this review is to highlight our current understanding of the molecular mechanisms underlying the regulation of vitamin D metabolism by FGF23, and to discuss the role of these mechanisms in physiology and pathophysiology. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.Apart from its phosphaturic action, the bone‐derived hormone fibroblast growth factor‐23 (FGF23) is also an essential regulator of vitamin D metabolism. The main target organ of FGF23 is the kidney, where FGF23 suppresses transcription of the key enzyme in vitamin D hormone  The latter finding turned out to be a seminal discovery with major impact in the field of bone and mineral homeostasis.Fibroblast growth factor‐23 (FGF23) was discovered in the year 2000 as a novel member of the fibroblast growth factor family. More or less at the same time, FGF23 was described in the murine brain in thalamic nuclei,1 whil isolated FGF23 as pathogenetic principle from rare human tumors causing tumor‐induced osteomalacia (TIO), another renal phosphate wasting disease. Injection of recombinant FGF23 into mice revealed two major actions of this factor: one action was the induction of renal phosphate wasting, resulting in hypophosphatemia, whereas the other was a distinct suppression of renal 1α‐hydroxylase (CYP27B1) mRNA expression. Renal 1α‐hydroxylase is the key enzyme for vitamin D hormone 2D) production. Later on, knockout experiments in mice revealed that Fgf23‐deficient mice were characterized by unleashed production of 1,25(OH)2D, causing hypercalcemia, hyperphosphatemia, ectopic calcifications, and early lethality. Similarly, loss‐of‐function mutations in FGF23 in humans cause tumoral calcinosis, a disease associated with elevated circulating 1,25(OH)2D levels, hypercalcemia, hyperphosphatemia, and progressive soft tissue calcifications from which the name of the disease was coined. Hence, in the absence of FGF23 the tight endocrine control of renal 1α‐hydroxylase transcription completely fails, leading to uncontrolled production of 1,25(OH)2D in mice and men. These findings have firmly established the essential role of FGF23 in vitamin D metabolism.The strong link between FGF23 and vitamin D metabolism became evident soon after the initial description of FGF23 as a putative phosphaturic hormone. Shimada and colleagues3 isolThe purpose of this review to highlight our current understanding of the molecular mechanisms underlying the regulation of vitamin D metabolism by FGF23, and to discuss the role of these mechanisms in physiology and pathophysiology. Vitamin D exists in two forms: cholecalciferol or vitamin D3 is derived from cholesterol in animals, whereas ergocalciferol or vitamin D2 is derived from ergosterol in fungi and protozoa. Both forms are referred to as vitamin D in this review. Depending on the species and the environmental conditions, vitamin D is either produced by a UVB‐mediated conversion of 7‐dehydrocholesterol in the skin, or it is taken up by ingestion of nutrients rich in vitamin D. Cutaneously synthesized or orally ingested vitamin D is then enzymatically converted to its most abundant circulating form, 25‐hydroxyvitamin D (25OHD), in the liver. This process is mediated by the high‐affinity cytochrome enzyme CYP2R1. 25(OH)D is released into the plasma and circulates in the blood mainly bound to the vitamin D–binding protein (DBP). However, in order for vitamin D to exert its biological effects, a subsequent hydroxylation step is necessary, which occurs in the kidney, the major source of circulating 1,25(OH)2D under physiological conditions. In renal proximal tubular epithelium, 1α‐hydroxylase converts 25(OH)D to its active form 1,25(OH)2D. Because 1,25(OH)2D is a lipophilic molecule it is able to cross cell membranes and to bind to its specific receptor, the vitamin D receptor (VDR), located in the cytoplasm and/or the nucleus of target cells. Ligand‐bound VDR forms a heterodimer with the retinoid X receptor (RXR). This complex can then bind to VDREs (vitamin D–responsive elements) to regulate gene transcription. The VDR is expressed in a wide range of tissues. Most mammalian cell types cells respond to 1,25(OH)2D exposure, with about 3% of the human genome regulated, directly or indirectly, by the vitamin D endocrine system. It is currently believed that all actions of the vitamin D endocrine system are mediated through the VDR.Vitamin D is an essential molecule for bone and mineral homeostasis in most vertebrates.8 Vita2D is the stimulation of intestinal absorption of calcium and phosphate. Overproduction of 1,25(OH)2D can lead to life‐threatening hypercalcemia and hyperphosphatemia. Therefore, synthesis and degradation of 1,25(OH)2D are tightly regulated. The major regulation of 1,25(OH)2D synthesis takes place at the level of 1α‐hydroxylation in the kidney, which is the rate‐limiting step in vitamin D hormone activation. In contrast, there is only a little regulation of 25‐hydroxylation in the liver. The major endocrine regulators of vitamin D metabolism are parathyroid hormone (PTH), FGF23, and 1,25(OH)2D itself. PTH is secreted in response to low plasma calcium and promotes 1,25(OH)2D synthesis by upregulating CYP27B1 expression in the kidney 2D production 2D is the regulation of the vitamin D inactivation pathway. Catabolism of 25(OH)D and 1,25(OH)2D is induced by 24‐hydroxylation (CYP24A1). All endocrine regulators of vitamin D metabolism regulate 1α–hydroxylase and 24‐hydroxylase expression in the kidney in a reciprocal manner. The stimulator of 1α–hydroxylase expression, PTH, suppresses CYP24A1 expression, whereas the suppressors of 1α–hydroxylase, FGF23 and 1,25(OH)2D, stimulate 24‐hydroxylase expression 2D intoxication. Hence, the regulation of circulating and intracellular concentrations of 1,25(OH)2D occurs at both the level of activation and detoxification pathways.The major physiological function of 1,25(OH)CYP24A1).13 All FGF23 is found in all vertebrates. Intact FGF23 circulating in the bloodstream is a 32‐kDa glycoprotein consisting of 227 amino acids. Only the intact form of FGF23 is biologically active. The main production site of this hormone are osteoblasts and osteocytes in bone.Within the gene family of fibroblast growth factors (FGFs), FGF23 belongs to the group of endocrine FGFs, together with FGF19 and FGF21.17 FGF In contrast to paracrine FGFs, endocrine FGFs lack a functional heparan sulfate‐binding domain, and require the presence of the transmembrane co‐receptor proteins αKlotho and βKlotho for high affinity binding to FGFRs. αKlotho was originally discovered as an anti‐aging protein. However, it was later found that αKlotho functions as co‐receptor for FGF23 signaling. A solid amount of data suggests that FGFR1c is the main FGFR mediating FGF23 signaling under physiological conditions. The b and c isoforms of FGFR1, FGFR2, and FGFR3 are generated by alternative splicing, and show major difference in ligand binding specificity.Canonical, paracrine FGFs contain a heparan sulfate‐binding site, which mediates binding of these FGFs to the local extracellular matrix, and also facilitates binding of paracrine FGFs to FGF receptors at the cell surface. All FGFs signal through four different FGF receptors (FGFRs), FGFR1, FGFR2, FGFR3, and FGFR4. FGF receptors are tyrosine kinase receptors, inducing phosphorylation of downstream molecules after ligand binding.19 In  FGF23 acts directly on proximal renal tubules. Binding of blood‐borne FGF23 to the basolateral αKlotho/FGFR1c complex downregulates the apical cell membrane abundance of the phosphate co‐transporters NaPi‐2a and NaPi‐2c via activation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2), serum/glucocorticoid‐regulated kinase‐1 (SGK1), and subsequent phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF)‐1. Phosphorylation of NHERF‐1 causes internalization and degradation of NaPi‐2a/c, thereby reducing tubular reabsorption of phosphate. Similar to FGF23, PTH, the other major phosphaturic hormone, downregulates renal tubular phosphate reabsorption by inducing NHERF‐1 phosphorylation via PTH receptor‐mediated activation of protein kinase A (PKA) and protein kinase C (PKC). Besides this phosphaturic action, FGF23 also suppresses the transcription of 1α‐hydroxylase in proximal renal tubules which is described in section Regulation of Renal Proximal Tubular 1,25(OH)2D Synthesis by FGF23.The kidney is the most important target organ of FGF23 under physiological conditions. In proximal renal tubules, FGF23 signaling regulates the membrane abundance of sodium‐phosphate co‐transporters.28 FGF Among other factors, 1,25(OH)2D, phosphate, PTH, and pro‐inflammatory cytokines have been postulated to play a crucial role in the regulation of bony FGF23 secretion. However, the molecular mechanisms underlying this regulation are still controversial. In vitro treatment of osteoblastic cells isolated from normal and uremic rats with 1,25(OH)2D increased Fgf23 transcriptional activity and FGF23 secretion. When the cells were treated with a medium containing only phosphate the effect was no longer present, suggesting that phosphate on its own cannot stimulate FGF23 production. This finding has been confirmed by other investigators. Kolek and colleagues observed that 1,25(OH)2D treatment of UMR‐106 osteoblast‐like cells induces a dose and time‐dependent increase in Fgf23 mRNA levels. Simultaneous treatment with a DNA transcription inhibitor and 1,25(OH)2D blunted the effect of 1,25(OH)2D on Fgf23 mRNA levels, suggesting that the regulation occurs at a transcriptional level.FGF23 secretion in osteoblasts and osteocytes is regulated at the transcriptional and posttranscriptional level by a plethora of local and systemic factors.33 Amo Moreover, these animals did not respond to treatment with 1,25(OH)2D, indicating that the VDR is necessary for regulation of FGF23. This was further corroborated by a recent study in mice with deletion of the Cyp27b1 gene. FGF23 levels were low in these animals, but exogenous treatment with calcitriol increased circulating FGF23.Indeed, in mice globally lacking VDR and in mice with a specific deletion of the VDR in chondrocytes plasma levels of FGF23 were lower than in wild‐type (WT) mice.38 Mor2D directly stimulates Fgf23 transcription or if an intermediary factor is necessary. Although Ito and colleagues were not able to identify VDREs in the promoter region of the mouse Fgf23 gene, six VDREs were identified in the human FGF23 gene that were capable of mediating direct transcriptional activation of a heterologous reporter gene by the VDR/1,25(OH)2D complex. Lee and colleagues deleted an enhancer region located directly upstream of the Fgf23 promoter in mice. This regulatory region in the murine Fgf23 promoter was identified in earlier chromatin immunoprecipitation sequencing (ChIP‐Seq) experiments. In mice with the proximal enhancer deletion the upregulation of Fgf23 transcription induced by 1,25(OH)2D injection as well as by an acute dietary phosphate challenge was lost, showing that this enhancer region participates in the 1,25(OH)2D‐mediated transcriptional regulation of Fgf23 in vivo. However, the 1,25(OH)2D‐induced upregulation of Fgf23 transcription may be indirect via other transcription factors, because ChIP‐Seq analysis in an osteocyte‐like cell line failed to provide direct evidence for VDR binding to regulatory regions in the Fgf23 locus.It is still unclear whether 1,25(OH)olleagues34 wer O‐glycosylation within the cleavage site protects FGF23 from cleavage during the secretory process, whereas phosphorylation near the cleavage site facilitates cleavage by preventing glycosylation. Because furin‐mediated cleavage occurs to some extent also during the normal secretion process, the balance between phosphorylation and glycosylation determines the relative amounts of intact and cleaved FGF23 secreted by osteoblasts and osteocytes. Several independent lines of evidence have suggested that extracellular phosphate is able to regulate posttranslational processing of FGF23. It is currently not known whether 1,25(OH)2D may not only be a transcriptional, but also a posttranslational regulator of FGF23 secretion.The secretion of bioactive, intact FGF23 is not only regulated at the transcriptional, but also at the posttranslational level by the balance between phosphorylation and glycosylation within or near the site of furin‐mediated cleavage.44 O‐gαKlotho‐deficient and Fgf23‐deficient mice, leading to uncontrolled production of 1,25(OH)2D. Similarly, humans with loss‐of‐function mutations in αKLOTHO or FGF23 are characterized by increased circulating 1,25(OH)2D. The sequelae of excessive 1,25(OH)2D synthesis are hypercalcemia, hyperphosphatemia, and extensive calcifications of soft tissues and blood vessels. The crucial pathophysiological role of excessive 1,25(OH)2D synthesis in diseases characterized by loss‐of‐function of FGF23/Klotho signaling is underscored by the fact that the phenotype of αKlotho‐deficient and Fgf23‐deficient mice can almost completely be rescued by concomitant ablation of vitamin D signaling.As mentioned above in the Introduction, the pivotal role of the FGF23‐Klotho signaling axis for the regulation of renal 1α‐hydroxylase is illustrated by the fact that despite hypercalcemia and suppressed PTH, 1α‐hydroxylase expression levels remain high in ent mice,48 leaαKlotho−/− and Fgf23−/− mice strongly suggest that FGF23 regulates 1α‐hydroxylase expression by an αKlotho‐dependent mechanism. In addition, it has been shown that a specific deletion of Fgfr1 in proximal renal tubules blunts the FGF23‐induced suppression of 1,25(OH)2D production in mice. Hence, there is good evidence that the FGF23‐induced suppression of 1α‐hydroxylase expression involves the FGFR1c/αKlotho receptor complex. However, FGFR3 and FGFR4 may also play some role, because it has been shown that ablation of Fgfr3 and Fgfr4 reduces the FGF23‐mediated suppression of renal 1α‐hydroxylase expression in Hyp mice, a model of excessive endogenous skeletal secretion of FGF23. In addition, the suppression of serum 1,25(OH)2D levels by injection of recombinant FGF23 was similar in kidney‐specific conditional Fgfr1 knockout mice compared with global Fgfr3−/− and Fgfr4−/− mutant mice. Proximal renal tubules express FGFR1, FGFR3, and FGFR4, but only a little FGFR2. Taken together, these findings suggest that FGF23 suppresses renal 1α‐hydroxylase expression by an αKlotho dependent signaling mechanism involving FGFR1, FGFR3 and FGFR4, with FGFR1c probably being the most important receptor under physiological conditions.Current knowledge about the molecular mechanisms involved in the FGF23‐mediated suppression of 1α‐hydroxylase transcription in proximal tubular epithelium is still fragmentary. The almost identical phenotypes of Hyp mice are associated with increased renal ERK1/2 signaling, and ERK1/2 inhibition increases 1,25(OH)2D production in Hyp mice in vivo. The signaling pathway further downstream of ERK1/2 is not known. Urakawa and colleagues established the transcription factor egr‐1 as one of the downstream targets of FGF23 signaling in the kidney. However, egr‐1 does not seem to be involved in the FGF23‐mediated regulation of renal 1α‐hydroxylase, because the FGF23‐mediated suppression of 1α‐hydroxylase mRNA expression was found to be similar in WT and egr‐1−/− mice. In addition, the FGF23‐mediated suppression of 1α‐hydroxylase mRNA abundance has been shown to be VDR‐independent, as evidenced by the fact that it is not blunted in VDR deficient mice.There is good evidence that the signaling events downstream of FGFRs involve ERK1/2 activation. The elevated circulating FGF23 levels in  in vivo.57 The has significantly advanced our understanding of the gene regulatory networks involved in the endocrine control of 1α‐hydroxylase transcription. ChIP‐Seq experiments in mice treated with recombinant FGF23 have shown that FGF23 controls the transcriptional activity of the 1α‐hydroxylase gene in the kidney through kidney‐specific regulatory elements located in introns of the neighboring Mettl21b gene. Moreover, deletion of the enhancer regions in the Mettl21b gene resulted in complete resistance to FGF23 treatment, providing firm evidence that FGF23 suppresses 1α‐hydroxylase gene transcription through these kidney‐specific enhancer sites. However, the transcription factor(s) binding to these enhancer regions, and, thus, the exact regulatory pathways are still unknown. In addition, it is important to note that mice with a deletion of all three enhancers in the Mettl21b gene do not recapitulate the phenotype of Fgf23‐deficient or αKlotho‐deficient mice which are characterized by a 1,25(OH)2D intoxication. Therefore, there are still gaps in our understanding of the suppressive effect of FGF23 signaling on renal 1α‐hydroxylase.At the genomic level, the work of Meyer and colleagues60 has However, it still awaits further clarification whether FGF23 signaling is able to directly regulate 24‐hydroxylase in the kidney in a VDR‐independent manner, or whether this regulation occurs via alterations in 1,25(OH)2D production and VDR signaling. Due to the fact that 1,25(OH)2D is a strong regulator of 24‐hydroxylase transcription, it is difficult to dissect direct and indirect effects of FGF23 signaling on 24‐hydroxylase in vivo. Experiments in VDR‐deficient mice suggested that the FGF23‐mediated regulation of 24‐hydroxylase is entirely VDR‐dependent, because treatment of these mice with recombinant FGF23 suppressed 1α‐hydroxylase, but failed to upregulate 24‐hydroxylase expression. In agreement with these data, the FGF23‐mediated induction of 24‐hydroxylase is completely lost in kidney‐specific 1α‐hydroxylase knockout mice unable to respond to FGF23 treatment with altered 1,25(OH)2D production. However, a potential pitfall in experiments with VDR‐deficient and 1α‐hydroxylase–deficient mice is that 24‐hydroxylase expression is profoundly suppressed in the absence of vitamin D signaling, which may mask acute effects of FGF23 on 24‐hydroxylase transcription.It is well established that the suppression of renal 1α‐hydroxylase induced by injection of recombinant FGF23 into WT mice is accompanied by an upregulation of 24‐hydroxylase mRNA expression in proximal renal tubules.28 How2D‐mediated induction of 24‐hydroxylase remained normal, suggesting that FGF23 and 1,25(OH)2D may have independent effects on Cyp24a1 expression. However, the exact mechanisms of the regulation of renal 24‐hydroxylase by FGF23 still remain obscure.Recent evidence in favor of an at least partially VDR independent effect of FGF23 signaling on 24‐hydroxylase expression came from a mouse model in which an enhancer region downstream of the 24‐hydroxylase gene was deleted. In mice with this deletion, the FGF23‐mediated induction of 24‐hydroxylase expression was blunted. In contrast, the 1,25(OH)pression.61 How2D produced in the kidney stimulates secretion of FGF23 in bone, which in turn downregulates 1,25(OH)2D production in the kidney 2D synthesis and degradation fail, resulting in inappropriately high circulating 1,25(OH)2D levels. It is evident that a downregulation of the PTH‐induced stimulation of renal 1α‐hydroxylase transcription cannot compensate for the lack of FGF23 signaling in these situations, highlighting the essential role of FGF23 signaling for the physiology of vitamin D metabolism. It is still unclear whether circulating FGF23 robustly suppresses PTH secretion tabolism.49 In  However, they profoundly differ in their effect on renal 1α‐hydroxylase expression. Teleological interpretations are always difficult to make and are often prone to error, but the principal physiological function of FGF23 may be to prevent hyperphosphatemia by increasing renal phosphate excretion and by indirectly reducing intestinal phosphate absorption through suppression of 1,25(OH)2D production, whereas that of PTH may be to counteract hypocalcemia. The prevention of chronic hyperphosphatemia is important for a healthy life, because this condition can lead to irreversible soft tissue calcifications with potentially serious side effects. PTH secretion is also stimulated by an increase in extracellular phosphate, but the stimulation of 1,25(OH)2D synthesis associated with a rise in PTH limits the capacity of PTH to deal with hyperphosphatemia. Therefore, the suppression of renal 1,25(OH)2D production makes FGF23 a more effective hormone for counteracting hyperphosphatemia compared with PTH. In situations with elevated PTH and subsequently increased 1,25(OH)2D to cope with hypocalcemia, FGF23 may fine‐tune the balance between bone resorption and intestinal absorption of calcium and phosphate by putting a brake on PTH‐stimulated 1,25(OH)2D production, possibly modulated by extracellular phosphate concentrations. Hence, from a systems biology point of view, it makes perfect sense to have two phosphaturic hormones with antagonistic effects on 1α‐hydroxylase expression in order to be able to cope with diverse disturbances in calcium and phosphate homeostasis.Why has nature equipped vertebrates with two phosphaturic hormones with antagonistic effects on 1α‐hydroxylase expression? Interestingly, there is good evidence from experimental and clinical studies that FGF23 and PTH interact in proximal renal tubules with regard to their phosphaturic actions in an additive or even synergistic manner.68 How2D synthesis aggravates the hypophosphatemia, because in addition to renal phosphate wasting, 1,25(OH)2D‐mediated intestinal absorption of phosphate is reduced. The major clinical problem in these patients are the skeletal manifestations of impaired bone mineralization due to hypophosphatemia. Therefore, the FGF23‐mediated impairment in vitamin D metabolism contributing to hypophosphatemia is an important part of the pathophysiology of renal phosphate‐wasting disorders.These useful physiological homeostatic mechanisms may turn into maladaptive processes in pathophysiological situations with chronically elevated FGF23. In renal phosphate‐wasting diseases characterized by inappropriately high circulating intact FGF23 such as X‐linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), or tumor‐induced osteomalacia (TIO), the FGF23‐mediated suppression of 1,25(OH) The reason for the elevated bony FGF23 secretion in CKD patients is still not entirely clear. It was previously thought that the rise in circulating FGF23 is driven by hyperphosphatemia due to the diminished capacity of the kidneys to excrete phosphate. However, clinical studies have clearly shown that hyperphosphatemia or increased PTH are not the drivers of FGF23 secretion in early stage CKD patients. Circulating intact FGF23 is a strong and independent risk factor for CKD progression, left ventricular hypertrophy, and mortality in CKD patients, and may directly promote left ventricular hypertrophy. It is clear that elevated FGF23 levels in CKD patients contribute to the suppression of 1,25(OH)2D production, which is already diminished due to the reduction in functional kidney mass. Whether this is good or bad is a matter of current controversy. Good aspects may be that low circulating 1,25(OH)2D levels help reduce intestinal phosphate absorption and do not further promote bony FGF23 secretion. However, bad aspects may be that low 1,25(OH)2D levels may compromise the immune system and may fail to suppress the elevated PTH secretion. This controversy has been fueled by a recent study showing that treatment of maintenance hemodialysis patients with secondary hyperparathyroidism and left ventricular hypertrophy with a vitamin D analog increased circulating intact FGF23 and aggravated left ventricular hypertrophy relative to a group treated with a calcimimetic that lowered circulating FGF23. These findings support the notion that treatment of CKD patients with vitamin D analogs at the expense of increased FGF23 secretion may have untoward effects on cardiovascular endpoints.In patients with chronic kidney disease (CKD) the blood concentrations of intact FGF23 rise stage‐dependently, and can reach very high levels in end‐stage renal disease.70 The2D production in proximal renal tubules by downregulating its synthesis through suppression of 1α‐hydroxylase expression, and activating degradation by increasing 24‐hydroxylase transcription. During recent years, major advances have been made toward a more complete understanding of the genomic mechanisms involved in the FGF23‐mediated regulation of vitamin D metabolism. However, there are still major gaps in our knowledge, as evidenced by the fact that the intracellular signaling cascades downstream of FGF receptors regulating transcription of 1α‐hydroxylase and 24‐hydroxylase in proximal renal tubules still remain undefined.The purpose of this review was to highlight the current knowledge about the molecular mechanisms underlying the regulation of vitamin D metabolism by FGF23 in health and disease. FGF23 is an essential regulator of vitamin D metabolism, suppressing 1,25(OH)NL and RGE have nothing to dislose.https://publons.com/publon/10.1002/jbm4.10558.The peer review history for this article is available at


Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27‐fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence‐independent control by adopting it for elongation control, achieving 8‐fold repression and 4‐fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis‐regulatory logic on CRISPRi‐targeted native genes.Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in  A regulatory control mechanism is developed based on the competitive binding of dCas9 to DNA by two guide RNAs. One represses a target gene (CRISPRi) and the second displaces the repressing dCas9 without interfering with the gene’s transcription. Integration can occur at individual promoters through the arrangement of DNA operators to which regulatory proteins bind and either recruit or interfere with transcriptional machinery  to overlapping positions that either recruit or block RNAP  directs dCas9 to the first position and a second derepressing sgRNA (sgD) directs dCas9 to a mutually exclusive second position that does not impact transcription Fig . We findet al, et al, E. coli constitutive promoter can be repressed by targeting the repressing sgRNA (sgR) to overlap the −10 or −35 σ70‐binding sites  and sgD (Vanillic acid‐inducible PVan) encoded on a p15A plasmid. The targeted constitutive promoter (PC1) was built based on the −35 to +1 core of PGate08 . From the same plasmid, dCas9 was expressed using the aTc‐inducible PTet promoter. A concentration of 1.25 nM aTc was used for all experiments and leads to approximately 500 dCas9 molecules per cell during exponential growth  and induced with aTc and either IPTG or Vanillic acid (Van) for 5.5 h . Fluorescence was then measured using flow cytometry. First, we compared the repression obtained by targeting the −10 (sgRP1) or −35 (sgRP2) positions of the promoter  of RFP fluorescence and ymax/ymin are the maximum/minimum measured values. Rx is the strength of the promoter driving the expression of sgR, measured as au of RFP fluorescence and Rκ is rescaled to be in the same units. This assumes that the binding of sgR to dCas9 to form RS is in the linear (unsaturated) regime. Equation P1 and sgRP2 induction curves ‐inducible promoter and sgDP5 from a Van‐inducible promoter  binding to the promoter to form DP,DK is the association constant between DS and P. The production of mRNA transcripts m from the promoter is described bymβ/Rα and mβ/Dα are the maximum transcription rates when either RS or DS are bound to the promoter. Solving for steady‐state yieldsOur model was then expanded to include derepression. Equation C2,As described for Equation P2  constant. The fit is shown as the heatmap coloration in Fig Dκ for sgD is similar to Rκ indicating that sgRP2 and sgDP5 have similar apparent binding strengths. Additionally, the observed αD of 1 is consistent with sgDP5 having little repressive effect on its own.This equation was then fit to the 2‐dimensional response of the promoter to the expression of sgR and sgD Fig . This fiet al . Starting with uninduced cells in exponential phase, we diluted cells into repressing conditions . Consistent with previous work, RFP fluorescence reduced at the rate of cellular division . This led to 47‐fold derepression within 2 h.We monitored the dynamics of repression and derepression for the promoter repression/derepression sgRNA pair sgRsion Fig . After 6et al, et al, et al, dCas9 can block transcription when it is directed to bind internally to a gene by physically interfering with the progression of RNAP Fig . Howeveret al, et al, O1) . sgRO1 was placed under the control of a Chol‐inducible PBetI promoter. It has been observed that elongation repression can be weaker with stronger promoters, presumably due to dislodgement of dCas9 by RNAP  to drive the transcription of the rfp reporter. When the promoter is strong (1 mM IPTG), we observed an 8‐fold repression of RFP upon the maximum induction of sgRO1 (10 mM Chol)  Fig . This lohol) Fig . While w Fig . Thured Fig . Represssion Fig . PreviouO2) and down‐stream (sgDO1) of the sgRO1 position  with the strongest induction of the rfp reporter (1 mM IPTG). Maximum expression of sgDO2 (100 µM Van) showed 4‐fold derepression . For each level of PTac induction, the relative expression during derepression was calculated as the ratio of the RFP fluorescence from the PTac‐only plasmid to the RFP fluorescence from the circuit plasmid with maximal sgRO1 and sgDO2 induction (10 mM Chol and 100 µM Van). From this experiment, we observed that derepression is invariant over a wider range of transcription rates than that observed for repression.The dependence of derepression on the PIPTG Fig . Similaret al, et al, et al, Van / PBetI). From this, we observed that the promoter repression/derepression circuit effectively responded to over 2 orders of magnitude of sgD/sgR ratios with a 50‐fold dynamic range  overrides the regulation imposed by sgR (A) Figs  and 3G. P2 and sgDP5) is capable of responding to transcriptional input ratios over two orders of magnitude  calculation, which if connected to a cooperative switch‐like output could act as a single neuron in a neural network  or other regulatory motifs  was used for all routine cloning. All genetic circuit measurements were done using E. coli K‐12 MG1655 * [F‐ λ‐ ilvG‐ rfb‐50 rph‐1 Δ(araCBAD) Δ(LacI)]  with the GC content set to 50%. Non‐linear fitting was completed with the Python scipy.optimize.curve_fit function.The random 100‐bp sequence within PAll growth was performed in 96‐well V‐bottom plates  at 1,000 rpm in a microplate shaker . The day before, individual colonies were inoculated into 150 µl EZ Rich media and Kan for overnight (16 h) growth at 37°C. The next day, cultures were diluted 200‐fold by adding 0.75 µl of overnight culture into 150 µl of EZ Rich media and Kan. After 2‐h growth, cultures were diluted 1,000‐fold into inducing conditions by adding 6 µL of culture into 198 µl fresh media and Kan and then 5 µl of that dilution into media with inducers and Kan. To induce dCas9, aTc was added to a final concentration of 1.25 nM in all experiments. Growth was performed for 5.5 h, after which samples were prepared for flow cytometry.https://github.com/cytoflow/cytoflow) was used for analysis, including gating. The geometric mean fluorescence is calculated for all cytometry distributions.Fluorescence characterization was performed using a BD LSR Fortessa flow cytometer with the HTS attachment . Samples were prepared by aliquoting 40 μl of cell culture into 160 μl of PBS containing 200 µg/ml Kan. All samples were run in standard mode at a flow rate of 2 μl/s. RFP fluorescence measurements were made using the green (561 nm) laser, and all data were derived from the PE‐Texas Red‐A channel (PMT voltage of 700 V). The FSC and SSC voltages were 650 and 270 V, respectively. At least 10,000 events were collected for each sample, and the Cytoflow Python package (Tac) reported in RFP fluorescence (au). This approach has been described previously  to the activities of the inducible promoter , and then resuspended in 300 µl of PBS and 2 mg/ml Kan and analyzed by cytometry. To transfer into de‐repressing conditions, 28 ml of culture was washed twice by centrifugation at 12°C at 4,300 g for 5 min. Then, the cultures were diluted to OD600 = 0.035 in 40 ml of media with 100 µM Van, 10 mM Choline, and 1.25 nM aTc.All growth was performed at 37°C and 350 rpm in an Innova44  with a 1‐inch throw. The day before the experiment, a starter culture was initiated by inoculating a colony into 2 ml EZ‐rich media with Kan in a 15‐ml test tube. The next day, this culture was diluted to an ODDAA and CAV conceived the study and designed the experiments. DAA performed the experiments and analyzed the data. DAA and CAV wrote the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.


Along with its capacity to reveal sites susceptible to undergo attacks simultaneously of nucleophilic and electrophilic types, another advantage of DD lies in being an orbital-free descriptor. That means it is based only upon total electron densities when written in its most accurate operational formula. This quality is not exclusive of DD because when Fukui functions are written in terms of electron densities instead of densities of frontier molecular orbitals, they become orbital-free descriptors too. Furthermore, the present work is an application of the generalized operational formula of the dual descriptor published in 2016 that takes into account any possible degeneracy in frontier molecular orbitals. As a proof about capabilities of DD, the possible sites for a favorable interaction between I2 with two organometallic compounds [Rh2(O2CCF3)4] and [(C8H11N2)Pt (CH3)] were correctly revealed by overlapping the biggest lobe for receiving nucleophilic attacks of one molecule with the biggest lobe for receiving electrophilic attacks of the other molecule, so allowing to predict the same coordination modes as experimentally known: linear “end–on” for the [(C8H11N2)Pt (CH3)]…I2, and bent “end–on” for the [Rh2(O2CCF3)4]…I2 interactions.The Janus–faced ligand behavior of diiodine (I Because it is a scalar field, it can represented as a 3D picture. Its main advantage lies in the fact that it provides more accurate information about local reactivities than the well–known Fukui functions. This fact is due to an arithmetic cancelation of relaxation terms when the electron density is expressed as a linear combination of molecular orbital densities  indicate the spin-multiplicity associated with each electron density, so that they are not powers. And subscripts  correspond to the total number of electrons associated to the respective electron density. Besides, p and q (the degrees of degeneracy of the LUMO and HOMO respectively), also indicate the number of hypothetical arriving electrons to the p–fold LUMO and leaving electrons from the q–fold HOMO.The so–called dual descriptor [DD or second order Fukui function] symbolized as oworkers ; 2006). ensities . Howeverensities . Hence, ensities  to obtaiensities , thus maσ*(I-I) orbital when interacting with the Pt atom of the [(C8H11N2)Pt (CH3)]. On the other hand, they also reported that diiodine behaves as an electron–donor towards the Rh2–core of [Rh2(O2CCF3)4] through the σ*(Rh-Rh) orbital. In the present work, the same conclusion concerning the double behavior of diiodine molecule is reached when using the operational formula given by Rogachev and Hoffmann  found thhttps://www.basissetexchange.org/), the def2–QZVPPD basis set Pt (CH3)], and [Rh2(O2CCF3)4] according to the following route section: M06L/GenECP NoSymm SCF= Int=. Notice that no symmetry restriction was set for all single-point calculations to freeze the Cartesian coordinates system, which is an essential procedure to build up the 3D pictures of scalar fields through the Gaussian software package. Pure d functions were added to the diiodine molecule through the option 5D.It is important to clarify that for the Pt and Rh complexes, coordinates for the optimized geometry were obtained from the data published by Rogachev and Hoffman. Geometry optimizations were done solely on the diiodine molecule in the gas phase. The route section for the optimization job was set as follows: M06L/GenECP SCF= Int = Guess = Huckel Opt=. Nevertheless, single point calculations in the gas phase were done on I–8 Hartree. The option Fermi speeds up the convergence of the SCF cycles by broadening the gap between virtual and occupied orbitals by means of fractionally occupied orbitals around the Fermi energy during the SCF cycles , 0.000006 for maximum displacement, and 0.000004 for RMS displacement with the VeryTight command. The CalcAll option in the Opt parenthesis computes the force constant at every point and with MaxCyc the maximum number of geometrical optimization steps was set to 300.For the integration process, setting the grid parameter to ultrafine  means thIn order to work with N = 106 , p = 1 and q = 2. The values of p and q follow Hund’s maximum multiplicity rule and take into consideration, respectively, the non-degenerate LUMO and doubly degenerate HOMO of the diiodine molecule , leading to the following definitive operational formula:Because 28 electrons per iodine atom are replaced with the pseudopotentials included in the def2-Q ZVPPD basis set, the total number of electrons dealt with is 50 (8H11N2)Pt (CH3)] and [Rh2(O2CCF3)4]. Two operational formulae of dual descriptor are inferred from 8H11N2)Pt (CH3)] complex (N = 160 − 60 = 100), and 28 electrons are also replaced by a pseudopotential for each Rh atom in the [Rh2(O2CCF3)4] complex (N = 310 − 2 ⋅ 28 = 254), the following operational formulae are obtained:An energetic threshold must be set by which to define degeneracy and hence the values for the variables p and q in 8H11N2)Pt (CH3)] complexFor the [(C2(O2CCF3)4] complexFor the  and [Rh2(O2CCF3)4] organometallic compounds through their operational formulae given by When quantum chemical calculations at the M06–L/def2–QZVPPD level of theory are performed for I8H11N2)Pt (CH3)] compound. The doubly degenerate frontier orbitals imply p = q = 2. On the contrary, there is no degeneracy of the frontier molecular orbitals of [Rh2(O2CCF3)4], in which case p and q equal 1. Energies of the nearest two virtual and two occupied molecular orbitals to the LUMO and HOMO, are quoted in 8H11N2)Pt (CH3)] and 2(O2CCF3)4]. Different cuts of the dual descriptor are displayed for the diiodine molecule in Following the 2% threshold criterium, a two-fold HOMO and two-fold LUMO are presented in the [(CThe opposite situation is depicted by 2) molecule exhibits an electrophilic behavior when facing [(C8H11N2)Pt (CH3)] leading to form a chemical bond of the type linear “end–on” coordination mode. But when I2 shows a nucleophilic behavior when facing [Rh2(O2CCF3)4] which leads to form a chemical bond of the type bent “end–on” coordination mode. This study agrees with Hoffmann’s theoretical evidence supported by MOT , NBO , and EDA .The operational formula of the dual descriptor (DD) adapted to molecular symmetry corresponds to an orbital–free local reactivity descriptor because there is no explicit dependence upon densities of fronter molecular orbitals, thus accomplishing with the essential philosophy of the Conceptual Density–Functional Theory: to explain all local reactivity in terms of electron density and not in terms of molecular orbitals. Furthermore, as an example, DD revealed that the diiodine . It is the responsibility of the authors to acquire the licenses, to follow any citation instructions requested by third-party rights holders, and cover any supplementary charges.