The key factors controlling the reactivity and stereoselectivity were systematically investigated by experimental and computational means and the energy profiles obtained provide a deeper insight into the mechanistic details of this reaction.A highly enantio‐ and diastereoselective [4+1] annulation between in situ generated ammonium ylides and  The 2,3‐dihydrobenzofuran skeleton represents an important motif in a variety of natural products and biologically active molecules.n+1] annulation reactions.A highly valuable approach to accessing chiral carbo‐ or heterocycles is the use of onium ylides for [n] annulation approaches relies on the use of in situ generated o‐quinone methides or analogous aza‐o‐quinone methides.o‐quinone methides 3 (precursor 1) for [4+1] annulation reactions with in situ generated carbonyl‐stabilised sulfur ylides 4  to access 2,3‐dihydrobenzofurans 5 was reported by Zhou and co‐workers  to access 2,3‐dihydrobenzofurans with control of both the relative and the absolute configuration. Besides the development of the first (highly) asymmetric ammonium ylide based protocol, which will overcome the less than satisfactory enantioselectivity observed so far when using chiral sulfur ylides, we also became interested in investigating this reaction in more detail by computational means to reveal the key factors in this ylide‐mediated [4+1] annulation reaction.Following our recent research focused on ammonium ylide mediated asymmetric annulation reactions6 a–c to investigate the influence of the nature of the carbonyl group on the reaction with o‐quinone methide precursor 1 a . Interestingly, the less‐stabilised amide‐based ammonium ylide derived from 6 b did not give any product at all (just the formation of unidentified side‐products), whereas the acetophenone‐based salt 6 c could be used to access 5 c in an excellent yield of 95 % and with almost complete trans diastereoselectivity under the optimised reaction conditions  as the chiral amine leaving group  and we also soon realised that Cinchona alkaloids with a free 9‐OH group (A2 and B1) allowed for higher enantioselectivities than the analogous O‐alkyl derivatives (see entries 4–9). Both enantiomers of trans‐5 c could readily be obtained by using either A2 or the pseudo‐enantiomeric B1, but it should be pointed out that A2 allowed for slightly higher enantiomeric ratios than B1. Based on our positive recent experience with compounds C,A2‐containing ammonium salt 6 c was still not complete after 20 h reaction time (which is in sharp contrast to the reaction with Me3N), longer reaction times of up to 3 days were necessary to achieve comparable yields for the enantioselective protocol . Overall, this obstacle makes a catalytic protocol unfeasible. However, it should be pointed out that the amine that is liberated during the reaction can easily be recovered after workup.Unfortunately, attempts to start from α‐bromoacetophenone and carry out in situ ammonium salt formation and ylide generation were not successful using either a catalytic or a stoichiometric amount of quinidine. As we mainly observed the decomposition of o‐quinone methides, we next explored the scope of this strategy. As can be seen in Scheme 1 and ammonium salts 6 were very well tolerated either in the racemic reaction  or in the asymmetric protocol. In all cases the trans products were obtained with almost exclusive diastereoselectivity and satisfactory isolated yields. The major exception in this regard was the asymmetric synthesis of the CF3‐containing product 5 h, which was achieved in a low yield of only 23 %, whereas the racemic protocol gave a yield of 88 %. We first reasoned that this could be due to a decomposition reaction between the product and the free Cinchona base that is liberated during the reaction. However, test reactions proved that the product 5 h is stable in the presence of A2 and therefore the exact reason for this striking difference remains unclear.Having identified the optimum conditions and the best‐suited chiral amine leaving group for the first highly asymmetric and high‐yielding formal [4+1] annulation of onium ylides to 5 s and t). It has been reported that the introduction of sterically demanding groups at this position leads to the preferred formation of the (Z)‐quinone methides, whereas unsubstituted derivatives mainly form E isomers.Z)‐quinone methide (or an E/Z mixture) in these two cases. We also tested one substrate 1 with a methyl group instead of the Ar1 substituent, which performed reasonably well in the racemic approach by giving 5 v in a yield of 99 % but with a slightly lower d.r. However, the enantioselectivity achieved in the asymmetric protocol turned out to be lower. We also carried out a gram‐scale experiment for the asymmetric synthesis of 5 i, which was thereby obtained with the same enantiopurity as in the experiment performed on the 0.1 mmol scale  and the o‐quinone methide generated form 1 a  due to the re‐aromatisation of the phenolic fragment. Two diastereomeric betaines can be formed during the addition step, trans‐ and cis‐A , but the latter is predicted to be formed through a lower free‐energy barrier  than trans‐A .Despite the sterically hindered character of the formed betaine, the initial addition step is slightly exothermic . In the case of trans‐A, the transition state (TS) of the ring closure lies lower in free energy than the addition TS, which indicates a non‐reversible initial addition step. For the cis pathway, the lower stability of cis‐A results in a partially reversible betaine formation. Indeed, the cis addition and cis elimination TSs are very similar in terms of free energy . These transition states lie, however, lower than the trans addition TS, and thus a cis selectivity is predicted, which is in disagreement with the observed high trans selectivity (trans/cis >95:5).Because the cyclisation occurs by an Scis‐2,3‐dihydrobenzofuran 5 c formation (kinetic selectivity) followed by an epimerisation of this latter. To test this hypothesis, we isolated cis‐5 c and subjected it to the reaction conditions. A slow isomerisation to the more stable trans‐5 c isomer was observed by NMR analysis and complete epimerisation was observed after 15 h (d.r.>95:5). Monitoring of the [4+1] annulation reaction over time revealed, however, that the >95:5 diastereomeric ratio in favour of the trans‐2,3‐dihydrobenzofuran is observed throughout the reaction, even in the first few minutes. Thus, given its slow kinetics, epimerisation of cis‐2,3‐dihydrobenzofuran cannot account by itself for the observed high trans selectivity.We reasoned that this contrast between computational predictions and experimental findings could be explained by a faster A by a deprotonation/protonation mechanism, such as previously observed by Aggarwal and co‐workers in sulfur ylide mediated cyclopropanation reactions.B . The observed high trans selectivity can thus be accounted for by selective cis‐A formation followed by rapid epimerisation into trans‐A, which then undergoes ring closure to give the trans‐2,3‐dihydrobenzofuran , ammonium salt 6 (1.2 equiv) and Cs2CO3 (2.5 equiv) were dissolved in DCM (15 mL mmol−11). The reaction mixture was stirred at room temperature for 3 days and afterwards extracted with DCM and brine. The combined organic phases were dried over Na2SO4, filtered and evaporated to dryness. The crude products were purified by column chromatography  gave the corresponding 2,3‐dihydrobenzofurans in the reported yields and enantiopurities.Product 5 c: Obtained as a white residue in a yield of 85 % on a 1 mmol scale (e.r.=99:1). [α]20D=−8.8 ; 1H NMR : δ=4.99 , 5.82 , 6.90 , 7.01–6.98 , 7.24–7.20 , 7.30–7.28 , 7.35–7.33 , 7.46 , 7.60 , 7.96 ppm ; 13C NMR : δ=51.0, 90.7, 110.1, 121.8, 125.5, 127.6, 128.3, 128.8, 129.0, 129.1, 129.5, 133.9, 134.6, 142.4, 159.2, 194.8 ppm; HRMS (ESI): m/z calcd for C21H16O2: 323.1043 [M+Na]+; found: 323.1040. The enantioselectivity was determined by HPLC =15.7, tminor =17.0 min).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.


Charge density fluctuations can be measured with orders of magnitude higher precision than previously accomplished using the G-band shift with charge. The two 2D intrinsic peaks can be associated with the “inner” and “outer” Raman scattering processes, with the counterintuitive assignment of the phonon closer to the K point in the KM direction (outer process) as the higher energy peak. Even low charge screening lifts the phonon Kohn anomaly near the K point for graphene encapsulated in hBN, and shifts the dominant intensity from the lower to the higher energy peak.Pristine graphene encapsulated in hexagonal boron nitride has transport properties rivalling suspended graphene, while being protected from contamination and mechanical damage. For high quality devices, it is important to avoid and monitor accidental doping and charge fluctuations. The 2D Raman double peak in intrinsic graphene can be used to optically determine charge density, with decreasing peak split corresponding to increasing charge density. We find strong correlations between the  The 2D Raman mode is of particular interest, as it involves two D band phonons with opposite, non-zero momenta. Despite the higher order process and non-zero momentum phonons, the 2D mode is the strongest Raman mode in single layer graphene. The double resonance (DR) mechanism2 explains why this higher order phonon mode is so strong; resonant virtual electron and hole scattering picks phonon q vectors that satisfy momentum and energy conservation. The DR process together with the strong K-point Kohn anomaly is why the 2D line provides so much information about the electronic and phonon dispersions around the K and K’ points. The phonon Kohn anomaly near K softens the D band phonon energies conically near K, resulting in a discontinuity of the derivative of the dispersion at K3. The slope of the phonon dispersion is a direct measure of the electron-phonon coupling (EPC) strength3. The DR and phonon dispersion are responsible for the shift in the 2D band energy with changing laser excitation energy4. Similarly, the strong dependence of the 2D mode on dielectric screening is readily observable by the shift in the 2D position for different environments, e.g., suspended graphene, graphene on SiO2, graphene on top of hexagonal boron nitride (hBN) or encapsulated in hBN, etc5. Larger dielectric screening6 will reduce the Fermi velocity, vF, so that the DR conditions will select larger phonon energy. The screening affects vF since electron energies are much higher than expected from a single particle picture, with a significant self-energy correction in a 2D material, in particular for free standing graphene near the charge neutrality point (CNP)9. The self-energy is decreasing as ε−1, where ε is the effective dielectric constant of the environment6. Charging the graphene layer will also renormalize and reduce the self-energy and therefore vF. For example, a study of gated graphene on hexagonal boron nitride (hBN) examined with scanning tunnelling spectroscopy demonstrated a change of vF from 1.3 to 1.05 × 106 m/s as the doping increased one order of magnitude from 2 × 1011 cm−2 to 2 × 1012 cm−2 9. Ultraclean suspended graphene with mobility reaching 106 cm2/Vs have shown vF reaching 3 × 106 m/s, measured by Shubnikov-de Haas oscillations7. Screening will also have an effect on the phonon dispersion, as it may decrease the strength of the EPC, and thereby weakening the Kohn anomaly at K, resulting in higher phonon energies with increased screening11. The reduced vF and reduced Kohn anomaly with screening are both expected to lead to a higher energy 2D peak. Hence, changes to the dispersion of the Dirac cone and the phonon branch can be experimentally deduced from the 2D peaks.Raman spectroscopy is a versatile experimental technique for exploring fundamental properties of graphene and characterizing properties such as layer thickness, defect density, charge and strain13. The vectors are found by translating the iso-electronic energy contour determined by half the laser energy at K, with a vector that puts the K iso-electronic energy contour tangentially in contact with the slightly smaller iso-energy contour (EL/2 − hcωD) at K’, shown in Fig. q vectors associated with the 2D Raman peak. While the DR Raman scattering is a two-dimensional process, the strongest contributions come from the high symmetry direction ΓK and KM, so that we can consider the one-dimensional case where electron (hole) scattering from ΓK to K’Γ followed by hole (electron) scattering back from K’Γ to ΓK are referred to as the outer process and the similar process between KM and MK’ as the inner process13, as shown in Fig. K was cylindrically symmetric, the 2D peak would yield two very different phonon frequencies from the inner and outer process since qi > qo where q is measured from the K point with cylindrical coordinates, shown in Fig. 12. Indeed, early in the study of graphene, a symmetric 2D peak for graphene on SiO2/Si was identified as a signature of a single layer14. Later studies showed that the intrinsic 2D line shape of single layer suspended graphene is asymmetric17, but reverts to a symmetric line shape when charge is added by field doping to 2 × 1011 cm−2, indistinguishable in shape from graphene on SiO218. Hence, we can deduce that there is not a perfect compensation of the phonon trigonal warping at low doping condition, and that the two peaks can reveal the behaviour of the inner and outer scattering processes, respectively. In addition, the energy upshift of the 2D peak is indicative of screening due to changes in the Fermi velocity, which has been pointed out previously20, but also due to changes in the phonon velocities12, addressed here.The DR method can be used for mapping out a contour of participating phonon wave-vectors21. The accidental doping in these samples is approximately ~1011 cm−2 or less as deduced from transport measurements22, more than an order of magnitude lower than for graphene on SiO2. We find strong correlations between the 2D peak splitting and the relative peak intensities and linewidths. The decrease of the 2D2 peak energy with charge screening is opposite to expectations. It follows from a crossover, shown in Fig. 11. This is a consequence of screening the Kohn anomaly at K. One has to carefully consider the ratio of the inner and outer Fermi velocities with the ratio of the inner and outer phonon velocities before determining if 2D1 is an inner or outer process. Here we present evidence to suggest that the lower energy 2D1 peak originates from the inner process, based on the calculated stronger intensity from the inner process in the low doping regime12, and linewidth behaviour. The analysis below is based on the assumption that increasing the charge also increases the screening. This assumption is supported by the reduction in Fermi velocity with increasing charge level, especially close to the charge neutrality point, attributed to screening the self-energy correction of the electronic energy dispersion9.Here we are considering the effect of very low doping levels in ultra-clean graphene, dielectrically screened by encapsulation in hBN. Encapsulated graphene possesses many of the superior transport properties of suspended graphene with the added advantage of being mechanically more robust and easier to fabricate2D1 and 2D2 can be used as a measure of charge doping two orders of magnitude lower than is possible to measure using the G band response to doping24.On a practical level, the peak split between 2D1 (blue), and the higher energy peak is denoted 2D2 (black). The 2D peaks measured at different locations have different 2D1 and 2D2 peak separations, linewidths, energies and intensities as shown in Fig. Raman data is collected from a graphene sample encapsulated in hBN. Examples of 2D spectra (λ = 532 nm) are shown in Fig. 25. Figure 2D1 (blue symbols) and 2D2 (black symbols) versus the corresponding G peak position where the different data sets are boxed with their respective laser colour. The red laser selects shorter q vectors through the DR mechanism and therefore has a significantly lower 2D energy than the green laser, as expected. Both data sets exhibit strain lines, indicating some compression26. We first consider Fig. 2D1 peak has a well-defined strain-induced slope of 1.7 ± 0.13, very close to the calculated value for uniaxial strain/compression in a random lattice direction26. The strain variation across the sample is on the order of ±0.1% which is small enough not to cause strain-induced peak-splitting27. The presence of the small strain variation will increase the linewidth only marginally26. The higher energy peak 2D2 does not fall along a single, well defined line. Instead, the data falls mainly in two groups: a lower 2D2− vs G, and a higher 2D2+ vs G. Despite there being two 2D2 groups, there is only one 2D1 line. (Supplementary Information (SI) Fig. 2D2− vs G and 2D2+ vs G are 2.2 ± 0.3 and 3.0 ± 0.4 respectively. The variation and range of the strain slopes matches earlier studies of graphene/hBN sandwiches28. For reference, 2D1 and 2D2 values for λ = 532 nm for freely suspended graphene has been included in Fig. 18.In order to evaluate the range of 2D spectra across the sample, we plot all the data measured on the same sample for two laser lines, λ = 532 nm and λ = 633 nm, in a 2D versus G diagram which reveals the contribution to the peak position due to strain or charge densityper se, but rather the effect of low charge doping on the graphene Raman peaks. The charge present is much lower than can be evaluated with help of a 2D versus G plot25, or by the G band linewidth or peak position24  to the right. Figure 2D1 and 2D2 band versus peak splitting, and Fig. 2D1 and 2D2 evolve as the splitting decreases. Figure Here we are not interested in the strain 11 cm−2 based on the back-gate voltage at the CNP. The charge variation is 0.4 × 1011 cm−2 given the residual conductivity at the CNP (see supporting information section 2). Comparing with our Raman data, which has a mean and standard deviation of −8.1 and 3.6 cm−1 for the 2D peak splitting, we find that for each cm−1 of 2D peak splitting, the doping changes by 0.11 × 1011 cm−2 making the 2D peak splitting a very sensitive probe of local charge density. Extrapolating linearly, we also find from this relationship that the CNP corresponds to a 2D peak splitting of 20.4 cm−1 (see supporting information section 3). While we know the mean and standard deviations for the doping in the sample used for transport measurements with high precision, and we know the mean and standard deviations for the 2D peak splitting for the sample used for Raman measurements, there is sample-to-sample variation which introduces uncertainty in our values for the slope of the linear correspondence and 2D peak splitting at CNP. We estimate a roughly 5% sample-to-sample variation, which translates into uncertainty in charge per wavenumber of 0.11 × 1011 ± 0.02 × 1011 cm−2 per cm−1, and 20.4 ± 1.1 cm−1 for the 2D peak splitting at the CNP.We establish a quantitative relationship between 2D peak splitting and doping by assuming a linear relationship between split and charge, and comparing means and standard deviations between the two. Transport measurements on a similarly prepared sample show that the mean doping level is 1.4 × 102/Si produces optical excitations of defect transitions in hBN and charge transfer to the graphene. In fact, light exposure during gating quickly returns the graphene to the charge neutrality point, undoing the gate doping29. The sign of the charge is also undetermined, but recent measurements30 on ultraclean samples have shown a symmetric electron and hole blue-shift of the 2D peak (single peak fitting) for charge density up to 2 × 1012 cm−2. Hence, the 2D peak response for holes and electron doping is indistinguishable on the Raman spectra at such low charge density. Early gated Raman measurements31 on graphene on SiO2 showed a weak difference between electron vs hole doping below 1012 cm−2 in contrast to the newer easurements30. This illustrates the need for excellent charge homogeneity to observe the low charge density effects achieved on suspended or hBN encapsulated graphene.While it would be advantageous to use electrostatic field gating to confirm, attempts to control the charge level via back-gating samples for optical measurements are complicated due to light-induced charge screening of the gate voltage for graphene on hBN. This is because light illumination on a gated graphene/hBN sample on SiO15, see Fig. q-dependent scattering matrix elements of the phonon intensity have shown that the inner process dominates despite the stronger vector nesting for the outer process13. Our results show the 2D1 peak with the strongest intensity at the lowest doping level. A higher 2D1 intensity has also been observed experimentally previously on both suspended18 and hBN encapsulated17 graphene. Based on the observed and predicted higher intensity for the inner process for pristine graphene, we tentatively assign the 2D1 peak as originating from the inner process, and show below how this assignment is supported by the results. Figure qi is larger than the outer vector qo, but the inner KΓ phonon dispersion is flatter than the steeper outer KM phonon branch32, as illustrated schematically in Fig. 2D1 as the inner process.Generally, a high density of vector nesting creates a stronger Raman peak. This would indicate that the outer process, with flatter parts of the trigonal warping of the iso-energy contour matched by the phonon contour, has a stronger contribution to the Raman spectra than the inner process2D1 to 2D2 with increased charge screening may be an effect of changing wave vectors and phonon energies that will affect both the scattering matrix terms in the numerator, as well as the denominator of the Raman intensity scattering expression12, which will change the relative strengths of the 2D1 and 2D2 peak due to quantum interference. Regardless of the cause for the intensity behaviour of the two 2D peaks, it is clear that added charge to graphene in hBN changes the dominant intensity from the 2D1 peak (inner) to the 2D2 peak (outer) with increasing charge.Intensity behaviour as a function of charge has not yet been explored theoretically. We suggest that the observed switch in intensity from 2D1 and 2D2 components and their linewidths is the ratio of the phonon velocity vph to the Fermi velocity vF for the inner and outer process. Using the DR mechanism and the simplifying assumption of perfectly linear electronic and phonon dispersions around the K and K’ points (but with different slopes for the inner and outer directions)13, and using vph ≪ vF, we have−1) at the K point (q = 0), γ is the imaginary part of the electronic self energy (eV), h is Planck’s constant, and c is the speed of light. The subscripts i and o refer to the inner and outer phonons, respectively. Equations 33, ∂ω/∂EL, of the 2D1 and 2D2 band for “pristine” and charged graphene and use Eqn.  of the inner branch, as expected11.It is counterintuitive that the rge Fig. , since iqi Fig. . The red2D1 and 2D2 are not indicative of the phonon lifetime, but rather determined by the electronic lifetime (imaginary part of the self-energy) and ratio of the phonon to electron velocity. However equation δqi,o. Phonons that fall within the blurred contour make a significant contribution to the 2D peak, and we will regard them as participatory phonons, and the blurred line as the participatory contour. The middle of the participatory contour is mapped almost perfectly to the triangularly warped iso-energy phonon contour with the “corners” pointing towards Γ rather than M. The width of the participatory contour in the KΓ direction (inner), δqi, has been calculated12 to be broader than the width in KM direction (outer), δqo, which provides a method for assigning 2D peaks to inner and outer processes. Using equations  The electronic self-energy is momentum dependent, and the momenta that contribute to the 2D1 and 2D2 peaks are different. Our observations are then consistent with an electronic self-energy that increases more greatly with charge for momenta that contribute to the 2D2 peak than momenta that contribute to the 2D1 peak. 2) The Fermi velocity decreases with charge less dramatically along the direction of the 2D2 process than the 2D1 process. If this is the case, then the 2D2 phonon velocity reduces by smaller amount than the 2D1 phonon velocity, and will result in a 2D2 linewidth that increases more rapidly with charge.Charge affects the 2D linewidth through two primary mechanisms: scattering and phonon velocity renormalization. Eqn.  shows thses Fig. . It is g2D1, has the highest intensity. The screening of the K-point Kohn anomaly with increasing charge has not yet been addressed experimentally on suspended graphene. A significant difference is the substantial decrease in the peak split at the CNP for λ = 633 nm, not observed for suspended graphene where the 2D peak-split stays nearly constant (~13 ± 3 cm−1) over a laser energy range from 1.5–2.7 eV18. This indicates that the dielectrically screened graphene is less well fit with a linear model than suspended graphene: The peak split EL, Berciaud et al.18 dismissed the theory of the peak split originating from the difference in the inner and outer 2D phonons. The phonon linewidth is expected to decrease with lower laser energy, since γeh ∝ EL, and Berciaud et al.18 observed an increasing linewidth for laser line energies below ~1.7 eV for suspended graphene. We note that it is possible that the predicted increase in phonon dispersion slope closer to the K point11, probed with lower laser energies, dominates over the decrease in electronic linewidth, γeh.The results presented here on hBN encapsulated graphene have both similarities and differences to suspended graphene. Both types of samples exhibit a decrease in the 2D double peak split with increased charge, and the lower energy peak, io, Eqn. , which iK-point Kohn anomaly with screening observed here is a sign of the weakened EPC3. Excitation of D band phonons by electrons is a fundamental bottleneck for ballistic transport at high fields35. Hence, it is possible that encapsulated graphene with low and uniform charge screening could have better transport properties at high fields than suspended graphene if the screening sufficiently reduces the strength of the EPC of the D phonons, without introducing other scattering mechanisms.The lifting of the 2D1 and 2D2 peak separation removes influence of strain, and reveals strong correlations between charge, peak intensities, peak positions and linewidths. The peak split can be used to estimate the charge density with sensitivity ~ 1010 cm−2 per cm−1 2D peak split, an improvement of two orders of magnitude compared to use of the G band Kohn anomaly at the Γ point. Hence, the 2D peak split could be very informative for detecting low amounts of charge, with the caveat that the peak split with charge behaviour is known for the particular substrate screening and laser line used. The method also reveals information about the origin of the double peak and the effect of increased charge, for example the intensity shifts from the inner to the outer phonon with increased charge. We associate the lower energy 2D1 peak with the so called inner process, and the higher energy 2D2 peak with the outer process. Even the low amount of charge puddling found in these samples is enough to significantly alter the strength of the D band Kohn anomaly at the K point.In conclusion, we have used Raman spectroscopy of graphene encapsulated in hBN to explore the intrinsic double peaked 2D phonon behaviour under low charge screening. The analysis of the 2D data as a function of 21. This particular sample did not have functioning edge contacts, but similar samples have a room temperature mobility of ~3.2 × 104 cm2/Vs.The encapsulated graphene sample was fabricated at Columbia University by Carlos Forsythe with their pick-and-place method that avoids direct contact between the pick-up polymer and graphene layer or the accompanying hBN surfaces that contact the grapheneEL = 2.33 eV) and red laser with wavelength of 633 nm (EL = 1.96 eV), are applied at 1 µm by 1 µm spacing with a laser spot of approximately 1µm2 with typical spectra shown in Fig. The collected 2D Raman spectral map is fitted with two Voigt peak functions at all different locations on the hBN encapsulated graphene. Different incident laser photon energies, green laser with wavelength of 532 nm (The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Info


The current data aimed to evaluate the performance of utilizing Al2SO4 reagent for activation of persulfate to treat landfill leachate. The data on chemical oxygen demand (COD), color, and NH3–H removals at different setting of the persulfate, Al2SO4 dosages, pH, and reaction time were collected using a central composite design (CCD) were measured to identify the optimum operating conditions. A total of 30 experiments were performed, the optimum conditions for S2O82−/Al2SO4 oxidation process was obtained. Quadratic models for chemical oxygen demand (COD), color, and NH3–H removals were significant with p-value < 0.0001. The experimental results were in agreement with the optimum results for COD and NH3–N removal rates to be 67%, 81%, and 48%, respectively). The results obtained in leachate treatment were compared with those from other treatment processes, such as S2O82− only and Al2SO4 only, to evaluate its effectiveness. The combined method  showed higher removal efficiency for COD, color, and NH3–N compared with other studied applications.Although landfilling is still the most suitable method for solid waste disposal, generation of large quantity of leachate is still considered as one of the main environmental problem. Efficient treatment of leachate is required prior to final discharge. Persulfate (S Specifications TableValue of the data•2SO4 to activate persulfate for leachate treatment.This article presents data on the performance of utilize Al•2SO4 alone and combined persulfate/Al2SO4.The data provides comparison of the treatment efficiencies between persulfate alone, Al•The data show the relationship between experimental factors and the responses statistically using mathematical models for COD colour and ammonia.•The optimum results can be useful for wide application on wastewater treatment.13-H removals at different setting of the persulfate, Al2SO4 dosages, pH, and reaction time  are presented in 3–N removals are presented in Eqs. 3–N, removal. 2SO4 and persulfate on COD color and NH3–N removal. 2SO4 and persulfate on COD color and NH3-N removal. 2SO4 and combined persulfate/Al2SO4 for COD, color and NH3–N removal.The data for general characteristics of landfill leachate used in this study are presented in . Furtherion time . The dat3–N removals are given in Eqs. 3), respectively.The second-order polynomial model for COD, color, and NH22.1Leachate samples were collected from the detention pond at Sungai Udang Landfill Site (SULS), Melaka, Malaysia. SULS has an area of 7 ha, receiving approximately 1200 t of municipal solid waste daily and start receiving waste at 1st of April 2015. In this study, the leachate samples were collected 6 times manually from February 2017 to Jun 2017 using 2 L plastic containers. The collected samples were immediately transported to the laboratory, characterized, and stored in cool room to 4 °C. The general characteristics of the leachate used in the study are presented in 2.22S2O8 M = 238 g/mol) and Aluminum sulfate (Al2SO4 342.15 g/mol) were used for advanced oxidation during the oxidation of leachate samples. Several dosages of S2O8 and Al2SO4 were gradually mixed with 100 mL of leachate samples to determine the optimum S2O82− and Al2SO4 dosage according to the efficiencies of COD, Color and NH3–N removal. Orbital Shaker (Luckham R100/TW Rotatable Shaker 340 mm × 245 mm) with at 200 rpm was used for samples shaking In the current study, Sodium persulfate  points and six runs at the center of the design 2.43–N, were immediately tested before and after each experiment. Leachate sample was shacked well analyzed. NH3–N concentration was measured by the Phenol Method No. (4500) using a UV–vis spectrophotometer at 640 nm with a light path of 1 cm or greater. pH was measured using a portable digital pH/Mv meter. COD concentration was determined by the open reflux method No. (5220). The test values are presented as the average of the three measurements, and the difference between the measurements of each value was less than 3%. The removal efficiencies of COD and NH3–N were obtained using the following Eq. iC and fC refer to the initial and final COD and NH3–N concentrations respectively.COD, color and NH


A systematic transmission electron microscope (TEM) study revealed a similar structural transition from amorphous to a distorted rutile structure in both cases, with a transition temperature of 700 °C for the NbO2 inside the MIM device and a slightly higher transition temperature of 750 °C for the reference NbO2 film. Quantitative composition analysis from electron energy loss spectroscopy (EELS) showed the stoichiometry of the nominal 15 nm NbO2 layer in the as-fabricated MIM device deviated from the target 1:2 ratio because of an interaction with the electrode materials, which was more prominent at elevated annealing temperature.Niobium dioxide can exhibit negative differential resistance (NDR) in metal-insulator-metal (MIM) devices, which has recently attracted significant interest for its potential applications as a highly non-linear selector element in emerging nonvolatile memory (NVM) and as a locally-active element in neuromorphic circuits. In order to further understand the processing of this material system, we studied the effect of thermal annealing on a 15 nm thick NbO At ~807 °C, NbO2 undergoes a reversible second-order phase transition resulting in a regular rutile structure 2 become a metallic conductor above 807 °C132 is thermodynamically stable within a narrow composition range with negligible deviation from the exact stoichiometry, NbO2-x sputtered films with well-controlled oxygen deficiency composition are reported to have superior TS2 accompanied the TS behavior2 films in two forms, a 180 nm thick film and a 15 nm thin film inside a nanoscale MIM device under thermal annealing in vacuum to emulate device processing conditions. High resolution transmission electron microscopy (HRTEM) and Scanning TEM associated electron energy loss spectroscopy (EELS) were performed to provide a systematic characterization of thermally induced crystallization in NbO2 films and the interaction between the NbO2 film and the electrodes.NbO2 film (180 nm). The amorphous structure in the niobium oxide film is shown by the uniform contrast of the NbO2 layer in the STEM image in 2 film was compared to a magnetron sputtered stoichiometric Nb2O5 film. Two-dimensional STEM/EELS mapping were collected from the entire niobium oxide layer in the NbO2 and Nb2O5 specimens, as illustrated by the cross-sectional STEM dark field (DF) image in the inset. Each spectrum in the spectrum image  was quantified based on integration of the Nb-M and O-K edges, with a standard power-law background subtraction and cross section calculation using the Hartree-Slater model162 and Nb2O5 were compared in 172O5 film . Similarly, the line profile in the films shows that the average of Nb at.% is 33.7 ± 0.4% in NbO2 , after removing a 10 nm surface with higher O composition due to exposure to the air after deposition. The EELS quantification results showed that both of the NbO2 and Nb2O5 films were close to the target composition. 2 film with aperiodic structure, which is confirmed by the diffuse ring shown in the FFT in the inset. There was no detectable structural or chemical variation in the film based on the combined TEM images and EELS quantification analysis.2 film 0 nm. The2, the as-fabricated 180 nm NbO2 film, as well as the 15 nm NbO2 film in the MIM device structure, were annealed at different temperatures up to 800 °C in vacuum to prevent further oxidization of NbO2192 film annealed between 700–800 °C. The amorphous structure in the 180 nm NbO2 film remained unchanged up to 700 °C. 2 film annealed at 700 °C for 20 min has a uniform contrast with no visible crystalline grains. This is confirmed by the selected area diffraction pattern (SAED) in 2 film annealed at 700 °C for 1 hr still remains as amorphous structure. 2 film at the atomic level. This suggests that 700 °C is certainly below the amorphous-to-crystalline transition temperature. A similar quantitative EELS analysis from NbO2 film annealed at 700 °C for 1 hr shows that the average of Nb at.% is 33.6 ± 0.5% in NbO2 film, consistent with that of the as-fabricated film. Figure 2 film started to crystallize after annealing at 750 °C. The nucleation starts on the surface of the film due to the higher density of defects and larger oxygen content near the surface. The grain size ranged from 10 nm to 100 nm. A SAED pattern from one of the larger grain shows a crystalline zone pattern of [103] from tetragonal NbO2 /W bottom electrode for the MIM device. The NbO2 layer had uniform contrast, indicating amorphous structure, which was distinct from metallic electrode materials including W, TiN, Pt, and Cr, all showing crystalline features from diffraction contrast. The thin layer of TiOx (3 nm) was deposited to enhance the adhesion between the top TiN and NbO2. High resolution TEM image in 2 and TiOx layers. All the layers maintained their chemical identity without obvious intermixing in the as-prepared state. The color elemental mappings processed from STEM/EELS SI in x, and NbO2 were consistent with the nominal deposition thicknesses. EELS analysis shows that there is no intermixing between Ti and Nb elements. After the TiOx layer was deposited on top of the NbO2, a thin oxygen rich layer formed on the top interface of NbO2 which probably was caused by the oxidation environment during the deposition of TiOx. The O map  inside the device stack222 crystallization in the MIM device provides a reference point for the calibration of the local temperature inside the device stack under Joule heating during electrically induced forming. The interaction between the switching oxide and the electrodes can change the local chemical composition during forming and subsequent operation, which provides important guidance for the design of selectors and the procedures used to form and operate them24This study provides a detailed structural and chemical analysis of the evolution of NbO2 thin films made by RF magnetron sputtering showed amorphous structure with uniform composition. EELS quantification analysis confirmed that the Nb at.% was 33.7 ± 0.4% in a 180 nm thick film (close to the stoichiometric NbO2 composition), and 35.3 ± 1% in a thin NbO2 (15 nm) layer in nanoscale MIM devices. In a cross-sectional TEM study, gradual crystallization to a distorted rutile structure was observed at 700-800 °C when thermally annealed in vacuum. Crystallization in the 180 nm NbO2 film started at 750 °C with some amorphous regions remaining, and full crystallization occurred upon annealing to 800 °C. Crystallization in the 15 nm NbO2 film within the nanoscale MIM devices started at 700 °C, with significant Ti mixed into the Nb oxide layer when annealed at 700–800 °C. These results suggest that crystallization and intermixing with TiN electrodes may occur in nano-scaled Nb oxide layers during operation of NbO2-based MIM switching devices, particularly during the high local temperature that typically occur during electrically induced forming step. Because Joule heating underpins the physical mechanism of the NDR behaviorIn summary, as-fabricated NbO2 or Nb2O5) were deposited by RF magnetron sputtering from either NbO2 or Nb2O5 targets. The deposition chamber base pressure was below 1 × 10−6 Torr and the substrates were heated to 450 °C during the deposition. For the 180 nm samples, the NbO2 blanket films were deposited directly onto silicon dioxide covered silicon substrates, whereas the 15 nm NbO2 thin films were deposited on a nanovia MIM device test platform. To fabricate the devices, a blanket W layer was first deposited by chemical vapor deposition (CVD) on SiO2 covered silicon wafer substrates. Then an insulating dielectric layer of silicon nitride/SiO2 was deposited on top of the wafers and was subsequently patterned and etched into contact holes of various diameter sizes by the photolithography process. To make the nanovia contacts to the bottom tungsten electrode, a titanium nitride (TiN) film was deposited inside the holes followed by chemical-mechanical polishing. The MIM devices were completed with a top electrode that consisted of 3 nm TiOx (for adhesion purpose), 6 nm TiN, 5 nm Pt, and 40 nm Cr that were deposited sequentially over the NbO2 film through a shadow mask. Thermal annealing of the samples was performed in a vacuum chamber at 1 × 10−6 Torr between 650 °C and 800 °C for different durations, i.e., 20 minutes and 1 hour. Cross-sectional TEM samples were prepared using a FEI Helios dual beam system. The TEM and EELS characterization were carried out by using a FEI Tecnai equipped with a Gatan GIF Quantum system. Two-dimensional STEM/EELS maps with EELS spectra in the 3rd dimension corresponding to each scanning pixel, called the spectrum image (SI), were collected with a probe size of 0.5 nm at a step resolution of 1 nm.Two forms of niobium oxide films .


A facile hydrogenation‐induced self‐assembly strategy to synthesize lithium hydride (LiH) nanosheets with a thickness of 2 nm that are uniformly distributed on graphene is reported and designed. Taking advantage of LiH nanosheets with high reactivity and a homogeneous distribution on graphene support as a nanoreactor, the confined chemical synthesis of oxygen‐free lithiated composites is effectively and efficiently realized. This method is limited, however, by the low and inhomogeneous loading of lithium in the composite, i.e., a loading capacity of 8.8 wt% for LiC6, which refers to the composition with maximal intercalation. The low loading‐concentration of nanostructured active materials would lead to significantly limited energy density of the whole composite, e.g., low hydrogen storage capacity as hydrogen storage material.One alternative method to synthesize and stabilize nanostructured materials is adopting templates to support their nucleation and growth during the self‐assembly process, which could further prevent the agglomeration and sintering of nanosized materials during practical applications.2BH3 and LiBH4 layers with a thickness of ≈4 nm, respectively, which present a layer‐by‐layer nanostructure, inheriting the structure of the LiH nanosheets homogeneously anchored on the graphene  give it the properties of an ideal nanoreactor for oxygen‐free lithiation of diverse composites to fabricate lithiated composites. As a proof of concept, by adopting LiH@G as a nanoreactor, we demonstrate a solid–liquid reaction and a solid–gas reaction to synthesize LiNHne Figure1. It iFigureAs illustrated in Figure 4H9Li and C6H12 are involved in the synthesis of LiH@G, density‐functional theory (DFT) calculations were conducted  and the FTIR results confirm the formation of LiBH4  and LiNH2BH3  with high purity, respectively.4/graphene hybrids (LiBH4@G) and LiNH2BH3/graphene hybrids (LiNH2BH3@G) exhibit a curved thin, flaky morphology, in which LiBH4 and LiNH2BH3 nanosheets are uniformly anchored on the graphene . No free LiBH4 and LiNH2BH3 nanosheets could be observed, and all of them remain uniformly anchored on graphene, even after long‐term ultrasonic dispersion for TEM characterization, which suggests the relatively strong interfacial interaction between LiBH4 or LiNH2BH3 and graphene. This therefore verifies the great potential of LiH@G as a smart nanoreactor for the oxygen‐free layer‐by‐layer assembly of lithiated composites toward enhanced performance in various fields.After the chemical reaction of LiH@G with B2H6 and NH3BH3 with high H2 capacity in LiH@G significantly upgrades the loading ratio of the as‐synthesized products and thereby improves the energy density in addition to the structural stability, e.g., a practical H2 capacity of 6.8 wt%, corresponding to ≈50 wt% for LiH in the composite, which could be increased to a practical H2 capacity of 12.8 wt%, corresponding to ≈69.1 wt% for LiBH4 in the composite. Simultaneously, the uniform distribution of LiH nanosheets and the steric confinement of graphene as structural support preserve the structural integrity and the nanosize effects of the as‐synthesized products. This unique sandwich configuration coupled with nanosize effects leads to significantly improved hydrogen storage performance based on a series of measurements. The mass spectra (MS) indicate that both the onset temperature (Tonset) and the peak temperature (Tpeak) are considerably reduced for LiH, LiNH2BH3, and LiBH4 anchored on graphene as compared to their bulk counterparts , with a peak temperature at ≈79 °C. Furthermore, a Tpeak of 346 °C was observed for LiBH4@G, 124 °C lower than that of its bulk counterpart, which achieves a practical hydrogen density of 12.8 wt% in the composite ). By comparison, the loading capacity of LiBH4 in templates is usually reported to be as low as 30 wt% or even less.4@G, owing to the tremendous reduction of particle size down to the nanometer scale.2. Since LiBH4@G hybrid displays the highest hydrogen capacity and favorable thermodynamics toward reversible hydrogen storage performance, LiBH4@G was further investigated in detail.When evaluated as hydrogen storage materials, the introduction of Berature Tonset and4@G were further investigated in comparison with the ball‐milled LiBH4 and the composite of LiBH4 and graphene (denoted as LiBH4/G). It could be clearly observed that only a capacity of less than 5 wt% hydrogen was released from the ball‐milled LiBH4 when heated at a temperature as high as 320 °C over a period of 300 min. Due to the catalytic effects of graphene on the hydrogen storage process in LiBH4,4/G with particle sizes of most materials larger than 400 nm and serious aggregation  exhibited improved dehydrogenation kinetics. Only 5.5 wt% hydrogen, however, could be released from LiBH4/G under the same conditions. By comparison, graphene‐supported LiBH4 nanolayers exhibited a fast dehydrogenation at only 280 °C with a capacity of ≈7.6 wt%, and the value further increased to ≈ 8.4 wt% and 9.5 wt% at the temperature of 300 and 320 °C, respectively. In particular, 9.7 wt% hydrogen could be rapidly released from LiBH4@G at a temperature as low as 340 °C in only 60 min. In order to gain better insight into the improvement of the dehydrogenation kinetics of LiBH4 nanolayers, the apparent activation energies (Ea) were quantitatively calculated based on the Arrhenius equation by fitting the experimental dehydriding kinetics at different temperatures . According to the slope of the linear plot of ln (dehydriding rate) versus 1/T , the Ea value for the dehydrogenation from LiBH4@G was determined to be 119.6 kJ mol−1, which is significantly lower than that reported for its bulk counterpart , corresponding to a capacity retention of ≈80%. The remarkable reversibility of LiBH4@G was further confirmed by the FTIR  and XRD  results, which demonstrates the regeneration of LiBH4 to its initial state. In addition, the formation of the stable compound Li2B12H12 in the rehydrogenated products was suppressed, which effectively prevents the loss of B during cyclic H2 release and uptake, and favors the stable cycling of LiBH4.4 nanoparticles, which is attributed to the reduction of particle size down to nanometer range.4@G is well preserved , during the dehydrogenation of LiBH4, owing to the reduction of particle size down to ≈4 nm, could circumvent the loss of B during reversible hydrogen storage. Furthermore, in the sandwich‐structured layer‐by‐layer structure between LiBH4 and graphene with its high surface area, graphene could act as a space barrier to effectively alleviate the particle growth and sintering of LiBH4 to a large extent during the thermal treatment involved in cycling  were fabricated via the hydrogenation of n‐butyllithium (n‐BuLi) in cyclohexane. In a typical synthesis, 2 mL n‐BuLi solution (2 m in cyclohexane) and 0.03 g graphene were dispersed in 40 mL cyclohexane in a pressure reactor vessel, and then the reaction was conducted under 50 atm H2 at 100 °C for 24 h with the aid of ultrasonication and stirring. After consecutive washing and centrifugation, the resulting composite was dried at room temperature under dynamic vacuum on a Schlenk line, denoted as LiH@G.4@GSynthesis of LiBH: As shown in Figure S18 (Supporting Information), LiBH4@G hybrids were synthesized by thermal treatment of 0.03 g LiH@G in a mixed B2H6/H2 atmosphere at 120 °C for 3 d. First, the reactor was loaded with LiH@G and a ball‐milled mixture of 0.6 g of ZnCl2 and LiBH4 with a molar ratio of 1:2, respectively, in the glove‐box. Subsequently, the mixed B2H6/H2 atmosphere was obtained from the decomposition (>90 °C) of a ball‐milled mixture of ZnCl2 and LiBH4. After the reaction of LiH@G with B2H6, the reactor was completely degassed via dynamic vacuum for 10 h at 80 °C to obtain LiBH4@G.2BH3@GSynthesis of LiNH: 0.04 g LiH@G was used as the nanoreactor, which was first dispersed in 5 mL tetrahydrofuran (THF) with the aid of sonication, followed by the slow addition of ammonia‐borane (0.0776 g) solution in THF (5 mL). Afterward, the mixture was stirred at room temperature for 3 h. Finally, LiNH2BH3@G hybrids were obtained by removing the solvent under dynamic vacuum.4/G CompositePreparation of LiBH: The bulk LiBH4/G composite with a weight ratio of 3:1 was prepared by ball‐milling technique using a planetary QM‐1SP2. The milling procedure with a ball‐to‐powder ratio of 50:1 and a milling speed of 350 rpm was carried out by alternating between 30 min of milling and 10 min of rest. All manipulation of materials was conducted in an argon‐filled glove box with H2O and O2 levels below 1 ppm to prevent contamination by air.Characterizations: Thermogravimetric analysis  was conducted on equipment connected to a mass spectrometer  using a heating rate of 5 °C min−1 under dynamic argon with a purge rate of 80 mL min−1. The phase composition of the powdery composite was analyzed by X‐ray diffraction  with Cu Kα radiation. To prevent any possible reactions between the sample and air during the XRD measurement, amorphous tape was used to cover the samples. Fourier transform infrared  analysis was adopted to verify the chemical bonding. During FTIR measurements (KBr pellets), a closed tube was used to load samples with KBr for measurement in an argon‐filled glove box. The morphology of the samples was determined using a field emission scanning electron microscope  and a transmission electron microscope . Elemental analysis was performed with an Elemen Tar Vario EL3 Elemental Analyser. The XPS were obtained on a Perkin Elmer PHI 5000C ESCA system equipped with a dual X‐ray source, in which an Mg Kα (1253.6 eV) anode and a hemispherical energy analyzer were used. The background pressure during data acquisition was maintained at <10−6 Pa, and measurements were performed at a pass energy of 93.90 eV. All binding energies were calibrated using contaminant carbon (C 1s = 284.6 eV).4@G was conducted at 320 °C under an initial H2 pressure of 100 atm for 5 h, and the desorption properties were determined at various temperatures under a hydrogen pressure below 0.01 atm.The hydrogen storage properties of the thus‐synthesized hydrogen storage materials were investigated on a Sieverts apparatus, denoted as a gas reaction controller . Rehydrogenation of LiBHSupplementaryClick here for additional data file.


Ka of 7–9 such as p‐nitrophenol, boric acid or a boronic acid. This provides an interesting alternative to an aldol reaction when combined with the straightforward addition of an alkyne to an aldehyde or ketone. The gold‐catalysed reaction of an electron‐deficient, sterically hindered propargylic alcohol with a boronic acid led to the formation of an unusually stable cyclic boron enolate.The regioselective gold‐catalysed hydration of propargylic alcohols to β‐hydroxy ketones can be achieved by diverting the gold‐catalysed Meyer–Schuster rearrangement through the addition of a protic additive with a p Propargylic alcohols are versatile compounds which can be used in a wide variety of gold‐catalysed transformations.o‐alkynlbenzeneboronic acids could be used as an extremely mild method to generate boron enolates 2 (Scheme A).3 would react with boronic acids to give half esters 4, which could undergo cyclisation to the corresponding boron enolate 5. In the event, these reactions led almost exclusively to the formation of Meyer–Schuster products 6. We observed, however, that a protic additive such as an alcohol or boronic acid served to accelerate the gold‐catalysed Meyer–Schuster rearrangement of propargylic alcohols 3 to enones 6 in a non‐polar organic solvent such as toluene (Scheme B and C).7a was observed as a significant by‐product in reactions of some secondary propargylic alcohols  could provide an interesting alternative to a traditional aldol reaction between a ketone and an aldehyde. This would circumvent the requirement to use low temperatures and stoichiometric reagents to achieve regioselective enolisation of the ketone, prior to addition of the aldehyde. Whilst isolated examples of gold‐ or mercury‐catalysed hydration reactions of propargylic alcohols have been described,We envisaged that a regioselective hydration of a propargylic alcohol to a ketone Scheme , D could7 directly (Scheme 5 which could yield 7via hydrolysis (path 2). We therefore set out to investigate in more detail the effect of the choice of protic additive on the product distribution of the reaction, with the aim of identifying conditions which would give the β‐hydroxy ketone 7a as the major product (Scheme 7a and the enone 6a were obtained (entry 1). Interestingly, the outcome of the reaction appeared to correlate reasonably well with the pKa of the protic additive used (entries 2–11). As the acidity of the additive increased  was used. However, as the acidity of the additive was increased further (entries 8–11), the product ratio reversed with enone 6a once again becoming the major product. This suggests that there is an optimum ‘pKa window’ in which the formation of 7a is favoured. With this information in hand, we went on to examine these conditions for the regioselective hydration of a selection of different propargylic alcohols . Hydration of propargylic alcohols 3g and 3h, to give aryl β‐hydroxy ketone 7g and branched alkyl ketone 7h, respectively, also proceeded smoothly. Initial attempts to achieve regioselective hydration of the fluorinated tertiary propargylic alcohols 3i and 3j were unsuccessful under the standard conditions as no reaction was observed. Higher conversions were seen upon heating the reaction mixture, but complex mixtures of products were observed with PNPOH as the additive. However, by using boric acid, the fluorinated ketones 7i and 7j were obtained in excellent yields from the direct hydration reaction, and in these cases no traces of the Meyer–Schuster rearrangement products 6i/6j were observed. Other tertiary propargylic alcohols such as 3k, derived from pinacolone, failed to give any β‐hydroxy ketone with the enone 6k being observed as the sole product  was accompanied by the formation of smaller quantities of the Meyer–Schuster rearrangement product . Interestingly, under the hydration reaction conditions, the conversion of the starting material 3 into both products 6 and 7 was considerably slower than the Meyer–Schuster rearrangement to give enone 6 in the presence of methanol as an additive.3) did not readily form the enone at all, whereas propargylic alcohols which contained groups able to stabilise a positive charge at the alcohol carbon  tended to form exclusively the enone 6. We therefore hypothesised that under these conditions, enone formation must proceed via a mechanism that involves an intermediate with substantial cationic character at the alcohol carbon. Coupled with the fact that the regioselective hydration reactions in Scheme 6 may take place via elimination of water from the desired β‐hydroxy ketone product 7. Resubmission of a purified sample of hydroxy ketone 7a to the reaction conditions  promotion of an alternative pathway leading directly to the β‐hydroxy ketone 7; (iii) Brønsted acid catalyst for the conversion of 7 into 6via elimination of water; and (iv) solubilisation of water in the non‐polar organic solvent.8, generated by gold‐catalysed addition of water or the protic additive9 takes place, followed by tautomerisation or hydrolysis to provide β‐hydroxy ketone 7. This then undergoes slow acid‐catalysed elimination of water . If ROH is not sufficiently acidic (MeOH), then intermediate 8 can evolve via proton transfer and then elimination of water, accompanied by cleavage of the carbon‐gold bond, to give allene 12, which undergoes tautomerisation or hydrolysis to provide 6. Again, the elimination step (11 to 12) must involve a transition state with considerably positive charge build up at the propargylic alcohol carbon, as enone formation was entirely suppressed in the case of strongly electron‐deficient propargylic alcohols (3i and 3j) The hypothesis that intermediate 8 should undergo protodemetallation with PNPOH is further supported by the fact that phenols  have been shown to be capable of protonating an sp2 aryl‐gold bond.3 (and indeed the β‐hydroxy ketone 7) can also act as a nucleophile and/or proton source in the mechanism.We believe that the protic additive5 as common intermediates in the formation of the enone 6 and β‐hydroxy ketone 7  acid additives. We have previously proposed cyclic enol boronates 7 Scheme . Enol bos Scheme .5p was confirmed by single crystal X‐ray crystallography  or electrophiles  in an attempt to either promote breakdown to the enone (6p) or β‐hydroxy ketone (7p), or to use 5p productively for new carbon‐carbon or carbon‐heteroatom bond formation were unsuccessful, leading to degradation or trace amounts of 7p only.The structure of y Figure .20 The s3p is consistent with the observations made during our study of the direct hydration reactions of other substrates (Scheme 3i–3j), in that the presence of the two carbon‐oxygen bonds adjacent to the propargylic alcohol is likely to prevent enone formation via destabilisation of the cationic transition state for the elimination of water from intermediate 11, hydroxy ketone 7  was added dropwise to a stirred solution of alkyne (1 equiv.) in dry THF (1 mL mmol−1) at −78 °C under an argon atmosphere. After 30 min aldehyde (1 equiv.) was added and the resulting solution was allowed to warm to room temperature and stirred overnight. The reaction was quenched with saturated aqueous NaHCO3 and the organic phase extracted with diethyl ether. The combined organic extracts were washed with brine, dried (MgSO4) and concentrated under vacuum. The residue was purified by column chromatography to give the propargylic alcohol.[3]Dec‐5‐yne‐4‐ol (3a): yield: 75%; IR (film): νmax=3331, 2958, 2933, 2873, 2231 cm−1; 1H NMR : δ=0.89 , 0.91 , 1.43 , 1.64 , 1.93 , 2.20 , 4.35 ; 13C NMR : δ=13.7, 13.9, 18.5, 18.6, 22.0, 30.9, 40.4, 62.6, 81.4, 85.6.3PAuNTf2]2PhMe (2 mol%) was added to a solution of propargylic alcohol (1 equiv.) and 4‐nitrophenol (1 equiv.) dissolved/suspended in toluene  and the solution stirred magnetically at room temperature until the starting material had disappeared by TLC (24–48 h). The reaction was quenched with aqueous NH4Cl and the organic phase extracted with Et2O. The combined organic phases were washed with brine, dried (MgSO4), concentrated under vacuum, and the crude product was purified by column chromatography to give the β‐hydroxy ketone.[Ph[21]7‐Hydroxydecan‐5‐one (7a): yield: 65%; IR (film): νmax=3429, 2958, 2932, 2873, 1704 cm−1; 1H NMR : δ=0.90 , 0.92 , 1.25–1.59 , 2.42 , 2.49 , 2.59 , 4.02–4.06 ; 13C NMR : δ=13.9, 14.1, 18.7, 22.3, 25.8, 38.6, 43.5, 49.0, 67.4, 212.7.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.


CLB2, the principal mitotic cyclin gene in Saccharomyces cerevisiae, and genes involved in fork stability and recombination. clb2Δ cells are not sensitive to HU, but the strong synergistic effect of clb2Δ with most genes tested indicates, unexpectedly, that CLB2 has an important role in DNA replication, in the stability and restart of stalled forks, and in pathways dependent on and independent of homologous recombination. Results indicate that CLB2 functions in parallel with the SGS1 helicase and EXO1 exonuclease to allow proper Rad51 recombination, but also regulates a combined Sgs1–Exo1 activity in a pathway dependent on Mec1 and Rad53 checkpoint protein kinases. The data argue that Mec1 regulates Clb2 to prevent a deleterious Sgs1–Exo1 activity at paused or stalled forks, whereas Rad53 checkpoint activation regulates Clb2 to allow a necessary Sgs1–Exo1 activity at stalled or collapsed forks. Altogether, this study indicates that Clb2 regulates the activity of numerous nucleases at single-stranded gaps created by DNA replication. A model is proposed for the function and regulation of Clb2 at stalled forks. These data provide new perspectives on the role of mitotic cyclins at the end of S phase.Paused or stalled replication forks are major threats to genome integrity; unraveling the complex pathways that contribute to fork stability and restart is crucial. Experimentally, fork stalling is induced by growing the cells in presence of hydroxyurea (HU), which depletes the pool of deoxynucleotide triphosphates (dNTPs) and slows down replication progression in yeast. Here, I report an epistasis analysis, based on sensitivity to HU, between  Mec1 and Rad53 prevent nucleolytic degradation at stalled forks by regulating nucleases and DNA-processing enzymes, including the exonuclease Exo1, responsible for fork collapse and double strand break (DSB) formation (rad53Δ cells in response to methyl methanesulfonate (MMS) and HU , secondary DNA structures, protein–DNA complexes, and gene transcription . Correctormation . Exo1 is) and HU . Howeverd by MMS . In addithe fork . Exo1 isin vitro and in vivo include the annealing of complementary strands of DNA, branch migration, regression of replication forks, and resolution of Holliday junctions that form at a collapsed replication fork or at recombinant structures  cells, after a short replication block, most forks resume progression. Prolonged stalling leads to fork inactivation and alternative pathways of fork restart, such as new origin firing  Saintig. It has e.g., Clb2 . Cdks interact with different cyclins throughout the cell cycle. Cyclins are essential activator of Cdks and are involved in the recruitment and selection of substrates. In budding yeast, six B-type cyclins (Clbs) associate with a single Cdk, Cdk1 (Cdc28), to drive S and M phase progression . Signifig., Clb2  or Clb6 ogenesis  and the ogenesis , the inhogenesis , and theactivity . In addiactivity . Cells wCLB2 and these genes , and are described in RAD52::TRP disruption was obtained using the vectors pMS21 cut with Bam1H (kindly provided by David Schild). The dna2-1 strain was obtained by sporulation of a diploid YLS37x ORD5350-5B (kindly gifted by Alain Nicolas), backcrossed five times with WT YLS37. The dna2-1 spores were identified by their thermo-sensitivity at 30°. For the quintuple mutants mec1Δsml1Δexo1Δsgs1Δclb2Δ, rad53Δsml1Δexo1Δsgs1Δclb2Δ, and rad53K227Asml1Δexo1Δ sgs1Δclb2Δ, to verify that mutant spores carry both the CLB2::TRP1 and the SML1::TRP1 markers, they were backcrossed with the isogenic WT type strain or alternative HIS+ strain. Diploids were then sporulated and a few tetrads were dissected and analyzed. The presence of tetrads showing a segregation (3/0 or 4/0) of the TRP1 marker indicated the presence of both the CLB2::TRP1 and the SML1::TRP1 disruption genes in the mutant spore. In addition, all spores were examined for the elongated phenotype of clb2Δ.Yeast strains used in this study are listed in Supplemental Material, Table S1 in File S1. All mutants with the same genotype were found to behave similarly unless specified.The number of independent mutants tested is indicated in Table S1 in Results section and in File S1.A variability of up to 10-fold of one or two mutants over a set of six or seven poor growth mutants was occasionally observed and is not unexpected given the high rates of mutations and chromosome rearrangements that occur in these mutants , and each dilution, starting with undiluted culture, was spotted out onto yeast extract peptone dextrose (YPD) plates with or without HU at the indicated concentrations. Of note, residual growth at the most concentrated cell densities was observed for all mutants, most likely owing to the time taken for HU to penetrate the cells. To allow an easier comparison of sensitivity, in some repeated experiments, if a mutant was observed to grow poorly, the cultures were concentrated (by spinning the culture at 3400 tr/min for 10 min and removing part of the YPD before suspension) to give approximately the same number of cells for all mutants. Alternatively, more cells were inoculated initially. Plates were incubated at 30° for 2–11 d. Images were taken starting on day 2 and every other day until growth ceased (up to 11 d).There are three types of genetic interactions, additive, epistatic, and synergistic. Additive interaction indicates that genes function in independent pathways. Deleterious effects of mutant alleles in independently functioning genes are expected to combine multiplicatively compared with WT (or mutant background). For example, a 20-fold effect from one gene mutation and a 10-fold effect from another gene mutation (both either increased or decreased with respect to WT at a given HU concentration) would be expected to result in a 200-fold effect compared with WT. The interaction is called epistatic if the phenotypic impact of the double mutation is less than expected . An epistatic interaction indicates that genes function within a common pathway or complex. Finally, a synergistic interaction, when the phenotypic impact of the double deletion is greater than predicted, is interpreted to reflect the existence of parallel (redundant or compensatory) pathways on a common substrate , whereas clb3Δ cells growth was similar to that of the WT strain. At lower HU concentrations, after 2–3 d of growth, the growth of the clb2Δ mutant was found to be similar to that of the WT cells  or parallel (synergistic interaction) pathways  and to infer regulatory hierarchies or functional complexes  is expected to be a mild growth defect on top of the effect of the associated gene deletion when plated at HU 200 mM, and no effect at lower HU concentrations.In order to investigate whether mitotic cyclins were involved in the replication process, the sensitivity to chronic exposure to HU for the WT, WT cells . This mimus81Δ mutants accumulate recombination intermediates during replication . The expectation for independent functions of these genes would be no effect on growth at 150 mM HU and a stronger growth inhibition at 200 mM HU, with at most a 10-fold drop in plating efficiency for the double mus81Δclb2Δ mutants. Surprisingly, though, whereas neither mus81Δ nor clb2Δ cells exhibited cell death at 200 mM HU, deleting CLB2 in mus81Δ cells resulted in complete cell death and strong growth inhibition at and below this concentration. Indeed, a decrease in plating efficiency of ∼50-fold at 150 mM HU and a decrease of at least 1000-fold at 200 mM HU was observed for the mus81Δclb2Δ mutants (File S1). Whereas mus81Δ cells continued to grow between 3 and 11 d at 200 mM HU  before cell death . A synergistic effect indicates that CLB2 and MUS81 function on a common substrate in parallel pathways, and the strength of the effect reflects an important role of CLB2 in this pathway . Thus, this result suggests that CLB2 has an important role in the dissolution of fork junctions or recombinant structures, when replication is slow, in the absence of the Mus81 resolvase. Alternatively, or in addition, the absence of Clb2 might lead to increased formation of fork junctions or recombinant structures that require processing by Mus81. This synergistic effect with mus81Δ supports the idea that Clb2 regulates the helicase Sgs1 , and sgs1Δ cells displayed cell death at elevated HU concentrations. The growth of the sgs1Δclb2Δ mutant appeared only slightly more inhibited than that of the single sgs1Δ mutant at HU concentrations ranging from 100 to 200 mM (File S1). However, at lower HU concentrations, at which sgs1Δ cells could form regular colonies (∼50 mM File S1), clb2Δ was clearly observed to mildly enhance the sensitivity and cell death of sgs1Δ cells by ∼20–50-fold (File S1). Thus, in contrast to the epistatic interaction observed in response to MMS . This, then, suggests that the synergistic effect of clb2Δ with sgs1Δ in response to HU is due to a role of Clb2 in preventing nuclease activity at stalled forks or in replisome stability or function in parallel with that of Sgs1.In addition to its role in resolving fork-like structures and Holliday junctions, the helicase Sgs1 prevents nuclease activity and ssDNA accumulation at the stalled fork to avoid fork collapse during replication . Since removing SRS2 favors the HR pathway, this synergistic effect suggests an important role of Clb2 in the HR pathway. Given that srs2Δ cells are not sensitive to HU, favoring the HR pathway, by itself, does not lead to HU sensitivity. This suggests that the absence of Clb2 leads to the formation of toxic recombinant structures when replication is slow. This result also supports the idea that Clb2 could regulate Sgs1 helicase in response to HU.The helicase Srs2 prevents HR during S phase (EXO1 shows a strong genetic interaction with SGS1 in the DSB repair pathway and in response to MMS (exo1Δ mutant resembles that of the WT at 200 mM HU (File S1). This suggests that Exo1 has a minor or no role during HU response in WT cells and is consistent with the inhibition of Exo1 activity by checkpoint function , consistent with the lack of effect of exo1Δ and the mild growth defect of clb2Δ.The exonuclease e to MMS . In respCLB2 could also regulate the associated gene, they show that in response to chronic exposure to HU, CLB2 functions in parallel with MUS81, SRS2, and SGS1. These results suggest that Clb2 is involved at stalled forks in the dissolution of fork junctions and the formation of recombinant structures, and support the idea that Clb2 could regulate Sgs1 in these two processes. In addition, Clb2 functions in parallel with Sgs1 in preventing nuclease activity at stalled forks or in replisome stability in response to HU.Thus, although these data do not exclude the possibility that SGS1, EXO1, and CLB2 (EXO1 did not affect cell viability in response to 200 mM HU (sgs1Δ cells to 60 mM HU (File S1). This synergistic effect suggests that Sgs1 and Exo1 function in parallel in response to HU, which is consistent with their redundant activity in DNA end resection. Deleting CLB2 greatly increased the HU sensitivity of double sgs1Δexo1Δ cells, as sgs1Δexo1Δclb2Δ mutants lost viability even at a very low HU concentration (20 mM). Growth of the triple mutant was impaired even on YPD plates without HU (File S1). This strong synergistic effect indicates that SGS1–EXO1 and CLB2 function in parallel pathways during replication and are essential for proper fork progression, when the dNTP pool is only mildly decreased. Given that Exo1 and Sgs1 have been shown to act redundantly in DNA end resection  allowed growth of rad51Δ cells but not rad51Δclb2Δ cells (File S1). Similarly, the rad52Δclb2Δ mutants were much more sensitive than the rad52Δ mutants. At 10 mM HU, a drop of ∼1000-fold (five mutants out of seven) or 100-fold (two mutants out of seven) in plating efficiency was observed for rad52Δclb2Δ mutants compared with rad52Δ mutants . At 15–18 mM HU, all the rad52Δclb2Δ mutants displayed a drop of at least 1000-fold in plating efficiency compared with rad52Δ mutants (File S1). This strong synergistic effect indicates that CLB2 has an important role during replication in parallel with RAD52 and RAD51 in response to HU. Given that ∼99% of rad52Δ cells and 65% of rad51Δ cells lose viability in the presence of a single chromosomal DSB , indicating correct chromosomes segregation, in line with the previous observation that chromosomes segregate correctly in the absence of DNA damage or after recovery from DNA damage in the clb2Δ mutant  deleting CLB2 in rad51Δsgs1Δ, rad51Δexo1Δ, or rad51Δexo1Δsgs1Δ increased cell death, indicating that the negative effect of clb2Δ in the rad51Δ context is not due (or not only due) to SGS1 and/or EXO1. In this study, the sensitivity of a rad51Δexo1Δsgs1Δclb2Δ mutant was compared with that of an exo1Δsgs1Δclb2Δ mutant in order to verify whether HR occurred in an exo1Δsgs1Δclb2Δ mutant. When plated at HU concentrations ranging from 8 to 15 mM, the four exo1Δsgs1Δclb2Δ mutants  displayed ∼10-fold differences in sensitivity in equal proportion (2/2). The data show that the rad51Δexo1Δsgs1Δclb2Δ mutant had comparable sensitivity to those of two exo1Δsgs1Δclb2Δ mutants out of the four, differing by less than five-fold in one experiment (File S1). However, the growth of the exo1Δsgs1Δclb2Δ mutants was much slower, in the presence of HU, , suggesting a very mild positive effect of deleting RAD51 in the exo1Δsgs1Δclb2Δ mutant. The two other exo1Δsgs1Δclb2Δ mutants were found to be more sensitive by ∼10–50-fold compared with the rad51Δexo1Δsgs1Δclb2Δ mutant at HU concentrations ranging from 8 to 15 mM (File S1), suggesting a positive effect of removing RAD51 in the exo1Δsgs1Δclb2Δ mutant. Thus, the results suggest that a small or very small number of toxic Rad51-dependent recombinant structures are formed in an sgs1Δexo1Δclb2Δ mutant and indicate that proper Rad51-dependent recombination does not occur in an sgs1Δexo1Δclb2Δ mutant. Since proper formation of the Rad51 nucleofilament requires adequate DNA resection, these results converge on the idea that Clb2 regulates DNA resection at stalled forks in a redundant pathway to Sgs1 and Exo1 so as to allow proper Rad51 recombination. Thus, these results indicate that Clb2 also functions in the HR pathway in the formation of recombinant structure at stalled forks, as already suggested by the srs2Δclb2Δ phenotype. These data further support the idea that Clb2 regulates ssDNA processing and nuclease activity at stalled forks during replication.In order to verify whether the deleterious effect of  context . As showperiment  and withdna-2-1 and rad27. Lagging strand DNA replication requires Okazaki fragment processing, including the cleavage of the displaced strand by the flap endonuclease FEN1 (Rad27 in yeast), before it is a few nucleotides long. If the flap extends to a longer length, it allows formation of secondary structure in the ssDNA, which inhibits FEN1 action, providing a switching mechanism for the processing of the flap between FEN1 and DNA2  to 10-fold (one out of eight) (File S1). In the rad27Δ mutant, deleting CLB2 increased the thermo-sensitivity ∼50-fold at 37° , indicating that Clb2 activity is in part responsible for cell death of the mec1Δ and rad53Δ mutants. A CLB2 deletion was also introduced into a strain expressing the rad53K227A allele, which retains WT RAD53-associated growth activity but is deficient for checkpoint function  by Mec1 and Rad53 checkpoint activation in response to HU. This is consistent with the report of Krishnan et al., which showed that Cdc28, in complex with Clb2, remains largely dephosphorylated and shows higher activity in mec1-1 cells in response to HU.Thus, whereas Clb2 activity is mildly beneficial in WT cells , it is dclb2∆ is hardly explained by a delay in cell cycle progression that would allow completion of replication, as Krishnan et al., showed that a mec1-1clb1Δclb2Δ mutant was only slightly delayed in spindle elongation compared with a mec1-1 mutant during HU treatment . The clb2Δ-induced rescue phenotype was observed in both mec1Δsgs1Δ and mec1Δexo1Δ cells. Surprisingly, similar to CLB2 deletion, deleting both SGS1 and EXO1 increased survival to HU of mec1Δ cells ∼100-fold at 3 mM HU (File S1). This synergistic effect suggests that Sgs1 and Exo1 have a redundant function that impairs the viability of mec1Δ cells. Since Sgs1 and Exo1 function redundantly in DNA end resection, presumably some fork collapse is due to deleterious and aberrant Sgs1 and Exo1 resection in the absence of Mec1. Thus, whereas Sgs1–Exo1 is necessary for viability in checkpoint-proficient cells in response to HU, in mec1Δ cells its activity is deleterious, suggesting that Mec1 inhibits deleterious Sgs1–Exo1 activity. This is consistent with the role of checkpoint proteins in preventing nuclease activity at stalled forks (sml1Δ) could not be tested in response to MMS, and have been revealed to be of interest in response to HU. The mec1Δsgs1Δexo1Δclb2Δ mutant was found to display sensitivity close to that of the mec1Δsgs1Δexo1Δ and mec1Δclb2Δ mutants, taking into account the growth on YPD of the mec1Δsgs1Δexo1Δclb2Δ mutant compared with mec1Δexo1Δsgs1Δ cells (File S1). Thus, deleting CLB2 did not improve further resistance to HU of a mec1Δsgs1Δexo1Δ mutant, as would be expected if CLB2 and SGS1–EXO1 functioned independently in mec1Δ cells. The fact that the effect is less than expected indicates an epistatic interaction and suggests that CLB2 and SGS1–EXO1 function in a common pathway in mec1Δ cells. The fact that the sensitivity of mec1Δsgs1Δexo1Δclb2Δ is close to that of mec1Δsgs1Δexo1Δ suggests that the clb2Δ-induced rescue phenotype in mec1Δ cells depends entirely on both SGS1 and EXO1. This result supports the idea that Clb2 could be regulating a complex formed by Sgs1 and Exo1. Alternatively, Sgs1 and Exo1 could regulate Clb2 activity.Combined deletions of /or EXO1 . In mec1ed forks . The quirad53Δ mutant, deleting EXO1 did not suppress HU sensitivity (SGS1 resulted in a mild improvement of rad53Δ viability to HU (File S1), although growth on YPD was impeded. Indeed, the rad53Δsgs1Δ mutant grew poorly , but its resistance was slightly improved when grown in the presence of HU compared with the rad53Δ mutant, suggesting that in the absence of Rad53, the activity of Sgs1 is important for growth but somewhat deleterious under conditions of reduced dNTPs. The clb2Δ-mediated sensitivity suppression was observed in both the rad53Δsgs1Δ and rad53Δexo1Δ contexts, although rescue of rad53Δsgs1Δ was slightly less efficient (File S1), suggesting that Sgs1 and Clb2 function in a common pathway and that Sgs1 is in part necessary for the clb2Δ-induced phenotype. The combined deletion of SGS1 and EXO1 suppressed further sensitivity of rad53Δ cells to HU compared with the single SGS1 deletion, but not to the extent of clb2Δ, even though growth was impeded on YPD (File S1). This rescue phenotype suggests that, in the absence of Rad53, the activity of Sgs–Exo1 is somewhat deleterious and is responsible for some fork collapse. This suggests that Rad53 inhibits deleterious Sgs1–Exo1 activity in WT cells. This is consistent with the role of checkpoint proteins in inhibiting nuclease activity at stalled forks.In the sitivity  , suggesting that both Sgs1 and Exo1 are necessary for the clb2Δ-associated phenotype in rad53Δ cells. This supports the idea that Clb2 regulates a complex formed by Sgs1 and Exo1, or combined Sgs1 and Exo1 activity. These results are hard to reconcile with the idea that Sgs1–Exo1 regulates Clb2, given that clb2Δ increases viability much more than exo1Δsgs1Δ, but the sgs1Δexo1Δclb2Δ-induced phenotype resembles the sgs1Δexo1Δ-induced and not the clb2Δ-induced phenotype in this context. However, it cannot be excluded that Clb2 could function downstream of and require Sgs1–Exo1 activity in the rad53Δ mutant.Most interestingly, the rad53Δ mutant, neither sgs1Δ nor sgs1Δexo1Δ suppressed sensitivity of the rad53K227A mutant, and the triple rad53K227Asgs1Δexo1Δ cell displayed sensitivity  comparable to that of rad53K227Asgs1Δ, rad53K227Aexo1Δ, or rad53K227A cells (File S1). Thus, although Sgs1 and Sgs1–Exo1 have a deleterious activity in the absence of Rad53, in the presence of Rad53 protein that lacks checkpoint activation but retains growth-associated activity, neither Sgs1 nor Sgs1–Exo1 has a deleterious activity in response to HU. This suggests that Rad53, independently of checkpoint activation, plays a part in preventing deleterious Sgs1 and Sgs1–Exo1 activity at stalled forks. Deleting CLB2 rescued both the rad53K227Asgs1Δ and rad53K227Aexo1Δ mutants, although less efficiently than the rad53K227A cells (File S1), suggesting that SGS1 and EXO1 are in part necessary for the clb2Δ-induced phenotype.Contrary to what was observed in the clb2Δ did not rescue the rad53K227Asgs1Δexo1Δ mutant. Instead, the rad53K227Asgs1Δexo1Δclb2Δ mutant exhibited HU sensitivity close to that of the rad53K227Asgs1Δexo1Δ and rad53K227A mutants, although growth on YPD was affected  activity of Sgs1–Exo1 occurs in the absence of Clb2 in the rad53K227A and rad53Δ mutants, and thus that Clb2 inhibits necessary Sgs1–Exo1 activity in the absence of Rad53 checkpoint activation. These results were interpreted to mean that, in WT cells, Rad53 checkpoint activation inhibits (or regulates) Clb2 to allow necessary Sgs1–Exo1 activity  and provide substrate specificity, the effect of deleting CLB2 in DNA replication  of Clb2 in the considered context, given that defects in fork processing and stalled fork structure differ in each context. The strong synergistic effect of clb2Δ with most genes tested reveals an important role for Clb2 in numerous pathways involved in the stability and restart of stalled forks during replication, by both HR-dependent and HR-independent mechanisms. This work indicates that Clb2 functions in the HR pathway at stalled forks during replication and at multiple steps  at collapsed forks . The expression of Clb2 starts at the end of S phase. Interestingly, late DNA replication regions have been defined as RSZs  Clb2–Cdk1 activity to prevent a deleterious Sgs1 and Exo1 activity, then Rad53 checkpoint activation inhibits (or regulates) Clb2–Cdk1 to allow Sgs1 and Exo1 activity. Since it could not be determined whether the phenotype of clb2Δ in mec1Δ cells was due to the absence of Mec1 or to a defect in checkpoint activation, it is possible that Mec1 could target Clb2–Cdk1 to prevent the nuclease activity of Sgs1–Exo1 at paused forks, prior to and independent of checkpoint activation. Interestingly, the Rad53-associated growth activity is also found to inhibit deleterious Sgs1–Exo1 activity, as suggested by the mild sgs1Δexo1Δ-induced rescue phenotype in rad53Δ cells and the lack of sgs1Δexo1Δ-induced rescue phenotype in rad53K227A (clb2Δ on growth on YPD in mec1Δexo1Δsgs1Δ and rad53K227Aexo1Δsgs1Δ mutant (both of which have Rad53 protein) contrasts with the mild positive effect in rad53Δexo1Δsgs1Δ mutant  [clb2Δ with rad52Δ, rad51Δ, sgs1Δ, and mec1Δ. Then, if replication is not able to restart and is stalled for a prolonged period, activated Mec1, leading to Rad53 activation, would inhibit (or regulate) Clb2 to permit Sgs1–Exo1 activity [clb2Δ with rad52Δ, rad51Δ, srs2Δ, sgs1Δexo1Δ, rad53Δ, and rad53K227A. Finally, Clb2–Cdk1 activity would regulate the dissolution of these recombinant structures by regulating Sgs1, consistent with the synergistic effect of clb2Δ with mus81Δ, and most likely by regulating the already identified Clb2 substrate Mus81 as well [One model for the roles of Clb2 and mitotic Cdk1 that accounts for these results could be that, at the end of the S phase, ssDNA is present at stalled forks in specific regions of the chromosome that are difficult to replicate, such as the RSZ . With ssIn summary, this work indicates important and numerous roles for Clb2 and mitotic cyclins in the replication process and suggests that mitotic cyclins play an essential part in the stability and restart of paused or stalled forks by modulating the activity of numerous nucleases at single-stranded gaps created by DNA replication. This work provides new perspectives on the role of mitotic cyclins in late replication by the end of S phase.www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300537/-/DC1.Supplemental material is available online at Click here for additional data file.


Ultrahigh rates realized by ALD‐made TiN. The symmetric full‐cell supercapacitors deliver a typical capacitance of 20.7 F cm−3 at a scan rate of 1 V s−1, and retain 4.3 F cm−3 at high rate of 100 V s−1. The devices can be charged and discharged for 20 000 cycles with negligible capacitance loss and with an ultralow self‐discharge current (≈1 μA). The resulting ultrafast‐charging TiN supercapacitors show good cyclic stability, ultrahigh power output, and energy storage capacities, which are comparable to existing carbon‐based microsupercapacitors.Metal nitrides have recently attracted increasing attention as suitable electrode materials for high‐performance supercapacitors mainly due to their excellent electrical conductivity. The conductivity of titanium nitride (TiN) varies between 4 × 10Figure2(OH)2CO3 nanowire precursor with an average diameter of about 100 nm was obtained by a standard hydrothermal method.2 film (20 and 50 nm) by atomic layer deposition (ALD) followed by removal of the cobaltous dihydroxycarbonate precursor in dilute acid. The resulting TiO2 nanotubes remain firmly attached to the metal substrate with quasi‐vertical alignment as shown in the scanning electron microscopy (SEM) images. Finally, the corn‐like TiN was obtained by annealing the TiO2 in dry ammonia atmosphere. The TiN samples derived from 20 and 50 nm ALD TiO2 are named as TiN‐20 and TiN‐50, respectively. The surface of the TiN‐20 nanowires is much rougher than that of TiN‐50 , which may be because of the limited deformation of thick TiO2 shell during annealing in ammonia. The length of corn‐like TiN is about 4 μm as seen in the cross‐sectional SEM image  experiments were performed at scan rates ranging from 0.1 to 100 V sFigure−1, TiN‐50 and TiN‐20 exhibit a volumetric capacitance of 20.7 and 10.0 F cm−3, respectively, higher than that of active carbon  and onion‐like carbon (1.3 F cm−3) under the same measurement condition.−1 due to the higher mass loading and denser nanostructure in the former. However, the TiN‐20 shows better capacitance retention  than the TiN‐50 when increasing the scan rate from 0.5 to 100 V s−1 . This may indicate a more facile ion transport for the TiN‐20 electrode.The surface of the TiN particles is highly accessible to the electrolyte, with very little impediment to ion transport, providing high capacitance even when operated at ultrahigh charge and discharge rates. τcharacteristic) is the time required to discharge the device with an efficiency of 63.2% and can be calculated by 1/(2πfcharacteristic).τcharacteristic values of TiN‐50 and TiN‐20 device are calculated to be 4 and 2 ms , in comparison with 400 ms for the AC supercapacitor and 0.3 ms for the electrolytic capacitor ,π factor was not included in previous work according to different definition of the time constant.To gain further insight into the mechanism of the ultrafast characteristic upon electron/ion transfer of TiN, the electrochemical impedance spectroscopy (EIS) was measured. The Nyquist plot shown in Figure e carbon  ms,π fac−3, which is comparable to the previous carbon‐based microsupercapacitors and lithium thin‐film batteries (10−3−10−2 Wh cm−3), and much higher than the previously demonstrated flexible TiN supercapacitor device (0.05 mWh cm−3).−3 at an energy density of 0.30 mWh cm−3, implies the capability of discharging within an extremely short time (14 ms). Detailed comparisons are listed in Table S1 (Supporting Information).A comparison of the different energy‐storage devices designed for high‐power device applications is presented in the Ragone plots . The data of TiN supercapacitors, as well as a commercial high‐energy lithium thin‐film battery 4 V/500 mAh) and high‐power aluminum electrolytic capacitor 3 V/300 mF) are included.00 mAh an0 mF are −1), the capacitance of TiN‐20 supercapacitor increases by about 5% after 20 000 cycles  to the device and monitoring the current while retaining the voltage. It is found from Figure Other significant factors in practical applications are leakage current and self‐discharge of the device.Umax. Basically, the voltage difference between the two terminals of the supercapacitor is recorded on an open circuit as a function of time. In general, most supercapacitors are operated in the range of Umax to 1/2 Umax, so the time required for the voltage across the supercapacitor changing from Umax to 1/2 Umax was measured. Figure The self‐discharge curves can be obtained immediately after charging the device to FigureIn consideration of practical use, combination of the supercapacitor devices is demonstrated for the purpose of increasing the operating voltage or output current. 2 on nanowire precursor followed by ammonia annealing. Such devices deliver high volumetric capacity, high energy and power densities, and negligible capacity loss after 20 000 cycles. A low leakage current (around 1 μA) and slow self‐discharge, two technically important features, are also obtained. We have also demonstrated series and parallel combinations of the TiN‐based supercapacitors. These ultrafast‐charging TiN‐based supercapacitors can directly power high‐power microelectronics, and their application may also be extended to flexible and wearable electronics.In summary, we have demonstrated a prototype of TiN‐based supercapacitors that exhibit ultrafast charging capability and slow self‐discharge without microconfiguration. The fabrication of the TiN electrodes is realized by ALD of TiOMaterial Synthesis: Corn‐like TiN nanostructures grown on different substrates  were prepared by the following process. Firstly, self‐supported Co2(OH)2CO3 nanorod arrays were prepared by a hydrothermal synthesis method following previous reports.3)2·6H2O, 4 mmol NH4F, and 10 mmol CO(NH2)2 in 60 mL distilled water. The resulting solution together with the substrates was transferred into Teflon‐lined stainless steel autoclave. The sealed autoclave was heated in an electric oven at 120 °C for 4 h, and then allowed to cool down to room temperature. The samples were then washed with distilled water and dried in furnace at 90 °C. For the second step, the samples were coated with a layer of 20 or 50 nm TiO2 by ALD (Beneq TFS200) using TiCl4 and water as the titanium and oxygen source, respectively. The deposited temperature was set at 120 °C. After the TiO2 shell coating, the samples were immersed in 1 mol L−1 HCl aqueous solution for 20 min to remove the Co2(OH)2CO3 template. Finally, the TiO2 shells were converted to corn‐like TiN by annealing in ammonia at 800 °C for 1 h. The mass loading of TiN was determined ≈1.01 and 2.30 mg cm−2 for TiN‐20 and TiN‐50, respectively.Supercapacitor Device Fabrication: The electrochemical performance of the corn‐like TiN was tested in CR2032 coin‐type symmetric supercapacitors, which were fabricated in atmosphere. The electrolyte was an organic electrolyte with 1 mol L−1 LiClO4 in nonaqueous acetonitrile solution. The positive and negative electrodes (ca. 1 cm2 in diameter) were separated by a porous nonwoven cloth separator and assembled into coin‐type symmetric cells.Characterization and Measurements: The microstructure and phase of samples were characterized by SEM (FEI SIRION), HRTEM (JEOL JEM‐2100F), XPS , and XRD . The electrochemical performance was investigated by CV, galvanostatic charge/discharge, and EIS measurements. CV and charge/discharge testing were performed on a CHI660C electrochemical workstation . EIS measurements were recorded on a VersaSTAT3 electrochemical workstation  over a frequency range of 100 kHz to 10 mHz with an amplitude of 10 mV at the open‐circuit potential. All experiments were performed at room temperature.Calculations: The volumetric capacitance (in F cm−3) was calculated using the voltammetric discharge integrated from the cyclic voltammogram over the whole potential range (1.4 V), according to the following equationQ (C) is the average charge, U (V) is the potential window, v is scan rate (V s−1), and V (cm3) is the synthesized TiN layer volume in the micrometer range.E, Wh cm−3) and power density  are calculated ast is the discharge time and C is volumetric capacitance calculated before.Energy density , is calculated usingThe characteristic time constant, 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.