The atomically sharp interface of this heterostructure exhibits clearly distinguished performance based on a series of characterizations. The oxygen evolution photoelectrocatalytic performance of the epitaxial M–S heterostructure is 2.5 times higher than that of pure MoS2 in microreactor, attributed to the efficient electron–hole separation and rapid charge transfer. This growth method provides a general strategy for fabricating seamless M–S lateral heterostructures by controllable doping heteroatoms. The M–S heterostructures show increased carrier migration rate and eliminated Fermi level pinning effect, contributing to their potential in devices and catalytic system.Accurate design of the 2D metal–semiconductor (M–S) heterostructure via the covalent combination of appropriate metallic and semiconducting materials is urgently needed for fabricating high‐performance nanodevices and enhancing catalytic performance. Hence, the lateral epitaxial growth of M–S Sn xMo1−xS2/MoS2 heterostructure is precisely prepared with in situ growth of metallic SnxMo1−xS2 at semiconductor MoS2 edge via one‐step chemical vapor deposition method. The oxygen evolution photoelectrocatalytic performance of the epitaxial M–S heterostructure is 2.5 times higher than that of pure MoS2 in microreactor, attributed to the efficient electron–hole separation and rapid charge transfer.The epitaxial growth of metal–semiconductor (M–S) Sn Nanostructured semiconductors have been normally used as photoelectric catalysts for solar hydrogen production, however, they suffered from the inferior characteristics, including unsatisfactory stability and photocorrosion under light illumination. The total energy conversion efficiency has been still far from ideal situation, in which the photoelectric catalysts should concurrently provide strong absorption range of sunlight, rapid charge transport, high density‐active sites, suitable band gap and band energy levels, large specific surface area, as well as they should have low cost and be environmental friendly.Excessive energy consumption leads to worsening environmental pollution and global warming. As a carrier of recyclable solar energy, sustainable production of hydrogen from water splitting has been intensively investigated.2, 3 Their ultrathin structure with large surface area provided great charge migration rate, strong optical absorption range with tunable electronic structure, and high mechanical flexibility. Interlayer stacking or intralayer splicing heterostructures have been demonstrated as effective strategy to precisely tailor the electronic properties. Thus, their photoelectrocatalytic performance for water splitting can be availably improved. For example, 2D semiconducting molybdenum disulfide (MoS2) was a favorable candidate with appropriate energy band gap (1.82 eV in monolayer) for solar absorption. More importantly, 2D MoS2 with high light transparency possessed outstanding electronic features, manifested as high carrier mobility and tunable charge‐carrier behavior. However, there are still several obstacles waiting to be addressed, such as photocorrosion, poor stability, and low photon‐conversion efficiency in visible light range, which have greatly limited their potential applications. These problems have been mainly due to their inefficient photogenerated electron–hole separation and low transportation efficiency, originating from the higher electron transfer barrier at the interface between the semiconductors and the electrodes. Hence, it is of great importance to optimize the interface structure to realize the fast electron–hole separation, minimize the photogenerated electron–hole recombination, and prolong the life of electron or hole for improving photoelectrocatalytic performance.2D transition metal dichalcogenides (TMDs) materials exhibited the quantum‐confined effect in the planar dimension with minimum interlayer interactions.6, 72‐based lateral heterointerfaces have been found to markedly enhance H2 or O2 production efficiency. A more elaborate design was the epitaxial M–S heterostructures, which were considered as intriguing building blocks for low‐power, high‐performance, and flexible electronic and optoelectronic devices. In the artificially in‐plane M–S heterostructures, the metallic 2D materials governed internal electrical transport, and the covalent connection replaced traditional physical electrode contact, which can dramatically improve charge injection, thus enhancing electrical performance. The covalently bonded contacts significantly lowered the Schottky barrier height, achieving ohmic characteristics by eliminating the Fermi level pinning effect. Furthermore, seamless stitching with metallic materials as contact electrodes can change the charge transfer kinetics and facilitate rapid electronic transmission across the interface. Hence, the direct in situ synthesis of seamless splicing 2D epitaxial M–S lateral heterostructure through covalent bond was highly desirable for effectively reducing the contact resistance and improving the carrier migration rate at interface to promote the photogenerated electron–hole separation in photoelectric catalytic system.The MoSficiency.18 A m In this work, we developed a heteroatom Sn doping engineering to prepare monolayer SnxMo1−xS2/MoS2 M–S epitaxial heterostructure via one‐pot CVD growth. In this method, seamlessly spliced metallic SnxMo1−xS2 was constructed at MoS2 edge by covalent bonds. A measurement system was developed in a homemade microreactor to explore the working mechanism of this 2D crystal in oxygen evolution reaction (OER)‐based photoelectrocatalysis. The M–S heterostructure crystal flakes were first transferred to Ti electrode on quartz glass sheets and then connected in the microreactor. The microreactor has following advantages: 1) A single tiny sample can be tested, and the specifically designed structure can be investigated and quantified. 2) Electron–hole instantaneous recombination resulted from mutual interference and hybridization materials can be effectively avoided in microreactor, thus improving light utilization rate per unit area. 3) The catalytic mechanism and charge transfer can be easily monitored in micromodel reaction for building the structure–activity relationship. This epitaxial M–S SnxMo1−xS2/MoS2 heterostructure showed an improved OER photoelectrocatalytic performance of 2.5 times, resulted from the efficient electron hole separation and rapid electron transfer with low resistance at the M–S interface. The in‐plane seamlessly splicing growth with 2D semiconductors and metallic alloys by covalent bonding provided a general strategy for the preparation of M–S epitaxial heterostructures. This artificial structure will be conducive to improving photoelectrocatalytic performance, accompanied by increased carrier migration rate.Chemical vapor deposition (CVD) is a powerful approach for preparing lateral epitaxial 2D heterostructures and stacking superlattices. However, the preparation of laterally epitaxial M–S heterostructures with atomically sharp interface through one‐pot or multi‐step methods has been still limited by the lattice mismatch and atomic compatibility.26 In  The electronic densities of states (DOS) of MoS2 and SnxMo1−xS2 calculated with the density functional theory (DFT) method are shown in Figure xMo1−xS2 confirmed that a slight Sn doping (≈2%) in MoS2 could cause a transition from typical semiconductor to metal. This result became the fundamental basis for the development of seamless splicing M–S SnxMo1−xS2/MoS2 heterostructures with in‐plane covalent bonds, which can be applied as high‐performance photoelectrocatalyst. The semiconductor and metallic alloy have the same phase structure and similar lattice constant, providing an efficient and ingenious design for seamless splicing M–S epitaxial heterostructure.It has been reported that heteroatom doping can induce the transition of electrical transport behavior of 2D materials, from semiconducting to metallic for M–S heterostructure construction.30 ThexMo1−xS2/MoS2 M–S epitaxial heterostructure was achieved via a facile CVD as shown in Figure 2 and SnxMo1−xS2 alloy deposited on SiO2/Si substrate separately was also prepared as a comparison . In a typical growth of SnxMo1−xS2/MoS2 heterostructure, SnO2 and MoO3 powders were selected as precursors, and NaCl was also mixed with precursors as catalyst to lower the growth temperature. The two precursors were spaced with an interval of 0.5 cm in the crucible, which prevented the volatile precursor from evaporating and depositing together on the SiO2/Si substrate, resulting in the growth of SnxMo1−xS2 alloy. As for the separate precursor powder, the upstream MoO3 reaches saturation vapor pressure first, the downstream volatile SnO2 reaches saturation vapor pressure later. The two kinds of powders are separated at a certain space, the growth sequences are separated in space and time. Thus, such spatial spacing results in that MoS2 are already preferred to grow in the central encounter. While SnS2 diffuses and is mixed with MoS2, this lead to SnxMo1−xS2 grow that the edge of MoS2 for SnxMo1−xS2/MoS2 heterostructure.The 2D SnxMo1−xS2/MoS2 heterostructure is displayed in Figure 2 and outside of Sn‐doped metallic SnxMo1−xS2 alloy. The color difference of atoms clearly distinguished the heterogeneous lateral interface of the structure. After growth, the flakes formed a concentric heterogeneous structure with two different color contrast . Raman and photoluminescence (PL) spectra were conducted to analyze MoS2 and metallic SnxMo1−xS2 . After Sn doping, new vibration peaks at 182.9, 220.4, and 346.1 cm‐1 were observed in Raman spectra for SnxMo1−xS2. The absence of PL excitation for SnxMo1−xS2 flake indicated their metallic behavior. Besides, in IDS–VG test, the IDS was linearly increased with VDS, independent with VG, which further demonstrated the metallic feature of SnxMo1−xS2 alloy . The results confirmed that small amount of Sn doping caused the transition of MoS2 from n‐type semiconductor to metallic SnxMo1−xS2.The atomic structure diagram of this Sn2 and SnS2 at the same temperature. MoS2 reached the saturated vapor pressure first because of the faster volatilization rate and then formed pure MoS2 core on the substrate. After that, the vapor pressure of SnS2 precursor existed and mixed with Mo to deposit SnxMo1−xS2 on MoS2 edges. The M–S heterostructures were formed by the resulting volatility variation and atomic compatibility. The growth mechanism can be clearly confirmed by the thermogravimetric analysis of SnS2 and MoS2 , in which Mo source was volatilized preferentially to Sn, and metal component of the vapor above the substrate was Mo and Sn atoms. Meanwhile, the edge of 2D monolayer MoS2 has many active suspension S–Mo bonds during growth, which are very conducive to further growth of SnxMo1−xS2 at edge of MoS2. Furthermore, SnxMo1−xS2 and MoS2 exist almost in the same lattice, this similar lattice can continue to in‐plane growth with no energy barriers and difficulties. Hence, we emphasized that this process should avoid external action during the growth of lateral M–S heterostructures, and maintain ideal conditions for 2H phase epitaxy by doping heteroatoms with minimal complication.The lateral M–S heterostructures were created along the growth sequence indicated in Figure  Under atomic force microscope (AFM), the epitaxial heterostructure exhibited uniform height at ≈0.79 nm monolayer with seamless connection , which was also consistent with the intrinsic characteristics of Fermi level in M–S heterostructures. Meanwhile, the stability test of this heterostructure was performed in air for 2 weeks  and SnxMo1−xS2/MoS2 flake was further proved to be a lateral epitaxial splicing heterogeneous structure.All the grown lateral M–S heterostructure crystals showed color contrast in core and edge regions with clear interface under optical microscope as shown in Figure 32 Und2 vibration peaks (E12g peak at 385.4 cm and the A1g at 405.9 cm) were observed in the core region from Figure 2 Raman peaks, three new vibration peaks  appeared at 182.9, 220.4, and 346.1 cm‐1, which were associated with the lattice disorder caused by Sn doping for SnxMo1−xS2. It showed that the Raman intensity ratio between LA(M) at J2 and E12g or A1g was inversely proportional to average interdefect/doping distance (LD). From the Raman spectra, the edge SnxMo1−xS2 showed a Raman intensity ratio of 0.237 for I(LA)/I(A1g) and 0.432 for I(LA)/I(E12g), indicating that the Sn doping concentration was at 2.8–4.0%. The crystal showed uniform MoS2 at A1g intensity in the whole regions . In the Figure  reports.38 In e Figure ,i. AfterxMo1−xS2/MoS2 structure was proved, the photoelectrocatalytic properties of the heterostructure was next investigated by evaluating its OER performance (4OH− + 4h+ = 2H2O + O2). To that end, we prepared devices with the schematics as depicted in Figure Von of 0.15 V and a current density Jon > 0.8 mA cm−2 (at 1.23 V vs RHE). These values suggested the heterostructure a significantly better photoanode than MoS2 nanosheets . In addition, the high Jon had a rapid response to visible light illumination with a response time <100 ms. With long time illumination up to 2 h, no detectable Jon decay (within our measurement error of 5%) was observed. The increase of Jon in the first 0.2 h may be attributed to the thermal effect of light illumination that led to an increased anode temperature. In contrast, MoS2 anode demonstrated a photocurrent decay by 70%, in agreement with the reported photocorrosion‐induced poor long‐term stability of 2D MoS2 crystals. As a control experiment, the OER performance of SnxMo1−xS2 was also evaluated. Despite of the conductive nature of the SnxMo1−xS2 nanosheet, the low Jon and high Von indicated that the doped Sn atoms made negligible effects on OER reactions.After the seamlessly spliced M–S SnxMo1−xS2/MoS2 heterostructure was a stable and photocatalytic anode compare to its two individual counterparts. To find out the mechanism, the time‐resolved transient absorption spectra (TAS) were applied to reveal the nature of the photocarrier dynamics of this heterostructure . As shown in Figure τ0 can be obtained by fitting the measured photocarrier dynamics using a single‐exponential function: ΔOD = c + A0 exp (–t/τ0), where ΔOD is the optical intensity, t is the time, and c and A0 are the fitting constants. SnxMo1−xS2/MoS2 heterostructure exhibited a τ0 of 10 ± 5 ps, faster than that of MoS2 28 ± 5 ps. Since τ0 was a direct measurement of the lifetime of photo generated carriers, it indicated an efficient and fast carrier relaxation process in the heterostructure from Figure Our results in Figure 2 in the heterostructure  measurements, in which the interface reaction dynamics of electrode was evaluated . The charge‐transfer resistance (Rct) values at the photoanode/electrolyte interface were summarized . Due to the presence of metallic SnxMo1−xS2 with low interface transport resistance, Rct of SnxMo1−xS2/MoS2 (≈17 Ω) was six orders of magnitude lower compared to that of MoS2. Based on above results, the improvement of the overall photoelectrocatalytic water splitting efficiency originated from 1) rapid electron transfer and 2) effective electron–hole separation, both of which benefited from M–S epitaxial heterostructures.We attributed such carrier behavior to the band structure of the heterostructure, which introduced an additional carrier decay channel for MoSe Figure . We recas Figure . When thxMo1−xS2/MoS2 heterostructures were accurately designed and prepared by sequential growth of monolayer Sn‐doping metallic SnxMo1−xS2 and semiconductor MoS2. The epitaxial growth of the metallic SnxMo1−xS2 at MoS2 interface can effectively eliminate the Fermi level pinning effect and minimize contact resistance. This M–S SnxMo1−xS2/MoS2 heterostructure is conductive to improving the photoelectrocatalytic performance compared to that of MoS2. The reason may be the significantly promoted separation of electron holes and effectively increased charge transfer. The accurate doping engineering method provided a general strategy for in situ growth of 2D metallic materials covalent bonding with appropriate 2D semiconductors for self‐assembling M–S epitaxial heterostructures. The seamless splicing of 2D metallic materials at the interface can have great application potentials in high‐performance devices by improving electron injection across the junction. They can also facilitate the applications in catalysis field for enhancing catalytic performance by rapid charge transfer with low‐contact resistances.In summary, a slight amount of heteroatom doping in 2D semiconductor can cause a transition from semiconductor to metal in electrical behavior. The epitaxial M–S SnxMo1−xS2/MoS2 epitaxial heterostructures were grown with a point‐to‐face metal‐source supply method by a simple atmospheric pressure CVD method. The SiO2/Si (270 nm SiO2) substrates were cleaned with piranha solution, isopropanol, and DI water, respectively. MoO3  and NaCl  were mixed and placed in front of the aluminum trioxide crucible, while SnO2  and NaCl (5 mg) were uniformly mixed and put behind the MoO3/NaCl mixed powder. The two growth powders were placed in the same crucible with space interval of 0.5 cm. The SiO2/Si substrate (size: 1 × 2 cm) was located above the powder on the crucible, which was placed in the middle of the furnace. Another crucible with sulfur powder  was put in upstream, ≈15 cm away from the middle of the furnace. The system was first ventilated with 100 sccm Ar for 10 min to remove other gases in the tube, and maintained an atmosphere with 100 sccm Ar. Then, the furnace was heated up to 695–710 °C for 40 min and maintained for 5 min, and then cooled to room temperature naturally.The M–S Sn2, 3 mg NaCl and 15 mg MoO3 powder were mixed together and placed in the middle of the tube furnace, and the temperature was maintained at 720 °C. For the growth of SnxMo1−xS2 alloy, the two precursors were mixed together with the same ratio (15 mg MoO3 and 28 mg SnO2), and 5 mg NaCl was mixed together at the same time, and the temperature was kept at 710 °C. The same ratio of two growth precursors was to ensure that the Sn doping concentration of SnxMo1−xS2 alloy was the same with that in the edge of SnxMo1−xS2/MoS2 epitaxial heterostructures.The growth methods were basically the same as above. For the growth of MoS2 was dissolved with NaOH. After being washed several times in water, the 2D transfer platform was used to transfer several samples to the Ti electrode. After baking, PMMA can be washed away with acetone and isopropanol.Working electrode was made of Ti electrode on quartz glass by photolithography and evaporation technology. The CVD‐grown 2D materials were spin‐coated with PMMA and SiO2 reference electrode and a Pt mesh counter electrode. The electrolyte was 0.5 m Na2SO4 with pH ≈ 6.8. The linear‐sweep voltammogram curves, I–t curves, and chronoamperometry measurements were performed under AM 1.5G illumination (100 mW cm−2) from a class AAA solar simulator . The linear‐sweep voltammogram curves were performed by scanning the potential from negative to positive direction at a scan rate of 0.005 V s−1. EIS was obtained on the workstation at 1.23 V voltage under light irradiation, with the frequency ranging from 0.1 Hz to 100 kHz.All the measurements were carried out on an electrochemical workstation  using a three‐electrode system, with Hg/HgClα source (λ = 1486.6 eV). The XRD was performed by Bruker D8 ADVANCE. The field‐effect transistors (FETs) were fabricated with In/Au as metal contacts directly on SiO2/Si substrate without transferring by standard photolithography and thermal evaporation. The dynamics of photocarriers of these samples were monitored by the time‐resolved TAS. A regenerative amplifier system  was used as the light source. The beam out of the amplifier was split into two beams: one was frequency doubled to get a beam of 400 nm as the pump beam; the other beam was focused into a sapphire plate to form the continuous white light with a range of 450–750 nm as the probe beam. The sub‐picosecond time delay between pump and probe beams was realized by a mechanic delay line . A spectrometer  was placed behind the homemade microscope to get the transient signals.The morphology of the samples was observed by optical microscopy (Nikon H600L). The SEM images were taken by Hitachi S‐4800 with 1–5 kV. The surface topography was examined with AFM (Bruker Dimension Icon AFM). The surface potential of these samples was quantitatively analyzed by KFPM (Bruker Dimension Icon AFM). Raman and PL spectra were performed using a WITec Alpha 300R spectrometer equipped with a 532 nm laser excitation and a CCD detector in a backscattering geometry. STEM images were acquired by using a JEOL 2100F microscope equipped with a cold field emission gun and double dodecaple correctors operated at 60 kV. The XPS was analyzed by the ESCALAB 250Xi XPS equipped with a monochromatic Al KThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The authors wish to make the following corrections to their paper :The heading “Conclusions” should be inserted before the final paragraph of the Discussion.The following citations in the ‘Discussion’ should be changed:[5] replaced by [7].[7] replaced by [13].[8] replaced by [14].[9] replaced by [5].[10] replaced by [15].In the Discussion, in the paragraph which begins with ‘Ultrasound assessment’, a new citation, [10], should be inserted after the following sentence: The ovulation results of the present study are similar to those of other studies.


The hydroxylation of nonreactive C−H bonds can be easily catalyzed by a variety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically. However, understanding the regio‐ and stereoselectivities of substrates hydroxylated by P450s remains a great challenge. Herein, we use a multi‐scale modeling approach to investigate the selectivity of testosterone (TES) and dihydrotestosterone (DHT) hydroxylation catalyzed by two important P450s, CYP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHT were predicted by dockings and molecular dynamics simulations, in which the experimentally identified sites of metabolism of TES/DHT can access to the catalytic center. The regio‐ and stereoselectivities of TES/DHT hydroxylation were further evaluated by quantum mechanical and ONIOM calculations. For CYP19A1, we found that sites 1β, 2β and 19 can access the catalytic center, with the intrinsic reactivity 2β>1β>19. However, our ONIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT, which is consistent with the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlying the selectivity of TES/DHT hydroxylation mediated by CYP3A4 and CYP19A1 and is helpful for understanding the selectivity of other substrates that are hydroxylated by P450s. Selective insertion: The results of a theoretical study on the selective hydroxylation of testosterone and dihydrotestosterone by CYP3A4 and CYP19A1 is reported (see scheme). The human cytochrome P450 superfamily includes 57 isoforms of heme‐enclosed enzymes that catalyze the redox reactions of a variety of endogenous and exogenous compounds.In the human body, hydroxylation reactions catalyzed by P450s are important in phase I metabolism.As an important case,In this work, we focus on the hydroxylation of two important steroid molecules, testosterone (TES) and dihydrotestosterone (DHT), catalyzed by CYP3A4 and CYP19A1. TES is a necessary male sex hormone and anabolic steroid.Here, we used a multi‐scale modeling approach, which involves molecular dockings, molecular dynamics (MD) simulations, quantum mechanics (QM) calculations, and quantum mechanics/molecular mechanics (QM/MM) calculations, to investigate the mechanism underlying the selective hydroxylation of TES and DHT by CYP3A4 and CYP19A1. The accessibility of each potential site of metabolism (SOM) to an enzyme's reaction center was evaluated by 100‐ns long MD simulations. The intrinsic reactivities of the potential SOMs were explored by using a prevailing truncated heme model. Together with the QM/MM calculations, our results unravel the mechanism underlying the selectivity of hydroxylation of TES and DHT by CYP3A4/CYP19A1.−1, respectively.There are several sites of TES that could potentially be oxidized by P450s. The experimentally determined oxidation sites of TES by CYP3A4 are 6β, 2β, 15β and 1β, and the corresponding reaction rates in the recombinant CYP3A4 enzyme are 83, 11, 5.0, and 4.8 minSince the crystal structure of the CYP3A4‐TES complex is not available, molecular docking was used to predict the initial binding mode of TES with CYP3A4. The experimental SOMs of TES are distributed in different rings, which means that multiple binding modes of TES could exist in the CYP3A4 active site. To consider the effect of active site residues on the binding of TES, multiple CYP3A4 crystal structures were used for docking.We have analyzed the root mean square fluctuation (RMSF) for each residue averaged over the 11 CYP3A4 crystal structures . The RMSFs for about 80 % of the residues are below 1.0 Å and those for the active site residues are mostly above 1.0 Å, especially for the residues of the F‐F′ helix, which show high RMSFs (ca. 4–5 Å). We therefore believe that the difference in the selected crystal structures is large enough for generating reasonable binding modes for TES to CYP3A4. By using these structures as the docking receptors, all experimental SOMs and the angular methyl groups were found to expose to the oxo moiety in the top ranked poses, named 6β‐4K9V, 2β‐3UA1, 1β‐4I4G, 15β‐4K9T, 18‐2V0M, and 19‐4D78. As shown in Figure MD simulations have been widely used in the study of P450 enzymes for a broad range of interests, including flexible conformational sampling,By applying the criterion, the accessibility for each site in the 12 CYP3A4 systems (corresponding to six docked TES and six superimposed DHT binding modes) and two CYP19A1 systems was evaluated as summarized in Figure For the 19‐4D78 system, the 6β site of TES is the dominant accessible site  contributes significantly to the intrinsic reactivity of hydroxylation,Even though it has been revealed by the QM calculations that sites 18 and 19 of TES are much less reactive, these sites can still be preferentially hydroxylated by different isoforms of P450s. The MD simulation results have been used to explain the selectivity of the above hydroxylation reactions. To further understand the mechanism underlying the selectivity of TES/DHT hydroxylation reactions catalyzed by CYP3A4 and CYP19A1, ONIOM calculations were performed. For CYP3A4, we chose the snapshots from the MD simulations of the 19‐4D78 and 6β‐4K9V systems to represent the binding modes of 17‐OH‐UP and 17‐OH‐DOWN, respectively. For CYP19A1, the representative snapshots from the MD simulations were used for the ONIOM calculations.−1, Figure −1, Figure In MD simulations, the 17‐OH group of TES/DHT was close to the upper region of the active site and was able to interact with the side chain of Glu308 Figure  A or the−1, Figure It should be noted that the 6β site of TES in the 17‐OH‐UP mode has a higher HAT barrier than that from the QM calculation , suggesting that the 6β site of TES is rather reactive and its activation barrier is hardly affected by the binding modes. The high reactivity of the 6β site has been predicted in the QM model and is considered as the main driving force for the selective hydroxylation of TES by CYP3A4.In the 17‐OH‐DOWN mode, TES/DHT is parallel to the heme plane. The activation barriers for sites 19, 6β and 8 of TES are 22.1, 10.8 and 16.6 kcal mol1 Figure  C, respe−1 . By using the spin natural orbital (SNO) analysis,Similar to the 19‐4D78‐DHT system, the 6β site of DHT could not access the oxo moiety either, since the “H‐oxo‐iron” angle was almost linear and unfavorable for shifting electrons to the substrate. For sites 19, 18 and 8, the activation barriers were 21.5, 23.5 and 25.8 kcal molThe hydroxylation of site 19 is the first step in CYP19A1 mediated TES aromatization, which finally produces estrogens.−1, Figure 3‐fold more efficient than CYP3A4 in generating the 19‐hydroxylated DHT.From the QM calculations, we learned that the 6β site of TES is most reactive. However, this site is far away from the Fe atom and even beyond the “6 Å” rule in the crystal structure of CYP19A1‐TES,For each site in DHT, the corresponding activation barrier is lower than that in TES Figures  E and F.CYP3A4 and CYP19A1 exhibited different regio‐ and stereoselectivities of hydroxylation towards TES/DHT. In this work, we examined these reactions in depth by taking into account the ligand reactivity and enzyme environment.By using the docking approach, we found that there exist two binding modes of CYP3A4‐TES; namely, 17‐OH‐UP and 17‐OH‐DOWN. MD simulations verified the stability of these binding modes. Furthermore, 6β was the most accessible site in the TES‐19‐4D78 system, with the accessibilities of sites 18 and 19 being significantly higher than other sites, especially in the DHT systems. From the QM calculations, we found that all the β sites are more reactive than the corresponding α sites, which explains the stereo‐selectivity of TES hydroxylation by CYP3A4. The results from the ONIOM calculations indicate that the hydroxylation of TES at the 6β site is independent of the binding mode, which explains why the 6β site is a major SOM. In the CYP3A4‐DHT systems, site 19 is more reactive in the 17‐OH‐UP binding mode than in 17‐OH‐DOWN because the activation barrier for site 19 is lower in 17‐OH‐UP.Although the crystal structure of CYP19A1‐TES has provided some clues for understanding the selectivity of hydroxylation, the structure still cannot be used to explain why the more reactive juxtaposition sites of TES/DHT are not the first choice of hydroxylation.In conclusion, our study has revealed the mechanism of the TES/DHT hydroxylation selectivity mediated by CYP3A4 and CYP19A1. Our results are also useful for understanding the regio‐ and stereoselectivities for the hydroxylation of other steroid molecules catalyzed by P450s.The initial model of CYP3A4 in complex with TES was obtained by molecular docking, which was executed by using GOLD Suite 5.2.The docking center was set on the Cpd I oxo atom and the space within 15 Å around the center was defined as the docking site. Fifty docked poses were generated for each docking and ranked by ChemScore with the scoring template parameterized for heme‐proteins.pmemd.cuda moduleAll the complexes obtained from the above step, that is, 12 CYP3A4‐TES/DHT and 1 CYP19A1‐TES/DHT, were subjected to 100‐ns MD simulations. The For each system, the energy optimization was carried out using the gradually decreased force constant restraints on the heavy atoms of the protein and ligand. The temperature of the system was then gradually raised from 0 K to 300 K in 80 ps using the NVT ensemble with the restraints the same as in energy minimization. An 80‐ps equilibration at 300 K without constraints on atoms was carried out using the NPT ensemble. Finally, the unrestrained production run was conducted for 100 ns using the NPT ensemble with a 2‐fs time step and 10‐Å cutoff for the nonbond interactions.−1 was adopted to control the temperature. The cpptraj moduleAll covalent bonds containing a hydrogen atom were constrained using the SHAKE algorithm.ϵ=4) for recovering the solvent effect.A truncated Compound I (Cpd I) modelpmemd module of Amber16. The geometrical optimization was then carried out with the ONIOM method. Thereafter, the TS structures for the SOMs were located using the flexible PES scanning. Each TS structure was further confirmed to have only one imaginary frequency. The procedure of preparing input files is similar to our previous study using the TAO package.S=1/2 and S=3/2 states for Cpd I mediated reactions are generally similar, only the doublet state of Cpd I was considered.chargesum module in TAO. The atoms in the QM region and the corresponding link atoms were optimized using the same level of theory and basis set as in the QM calculations. The SP energy was calculated using BS2 without applying any implicit solvent model and corrected using the same B3LYP‐D3 functional as in the QM calculations. For the molecular mechanics part, the AMBER force field was employed. Only the mechanical embedding scheme was considered in the geometry optimizations and single‐point energy calculations.For TES and DHT, the potential SOMs are located on the A, B, D rings and the angular methyl groups. In this study, QM/MM calculations were carried out to evaluate the activation barriers of potential SOMs in the protein environment. The QM/MM calculations were accomplished using the ONIOM method in Gaussian 09.The ONIOM calculations were performed using six representative MD snapshots, including four snapshots for CYP3A4; namely, TES‐19‐4D78, DHT‐19‐4D78, TES‐6β‐4K9V and DHT‐6β‐4K9V, and two snapshots for CYP19A1 complexes, for predicting the activation barriers of potential SOMs. For each system, the atoms of the nearest residues in the bottom area of the binding site together with the substrate and some other residues were selected as the QM region atoms. For 19‐4D78, atoms forming the hydrogen‐bond network TES‐water‐Glu308 and DHT‐water‐Leu482, together with Ser119, Ala305, Thr309, Ile369 and Ala370, were included in the QM region. For 6β‐4K9V, the atoms of Ser119, Thr282, Ala305, Ala370 and Leu482 were included in the QM regions. For the QM regions of the CYP19A1 systems, Ile132 and Thr310 were treated as the bottom area residues and Asp309, Val370, Met374 and Leu477 were included.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


We here report the development of a novel, user‐friendly, fully automated, and good manufacturing practice (GMP) compliant low‐pressure synthesis module for 11C‐carbonylation reactions using [11C]CO. In this synthesis module, [11C]CO was reliably prepared from cyclotron‐produced [11C]carbon dioxide ([11C]CO2) by reduction over heated molybdenum and delivered to the reaction vessel within 7 min after end of bombardment, with an overall radiochemical yield (RCY) of 71%. [11C]AZ13198083, a histamine type‐3 receptor ligand, was used as a model compound to assess the functionality of the radiochemistry module. At full batch production conditions , our newly developed low‐pressure 11C‐carbonylation apparatus enabled us to prepare [11C]AZ13198083 in an isolated radioactivity of 8540 ± 1400 MBq (n = 3). The radiochemical purity of each of the final formulated batches exceeded 99%, and all other quality control tests results conformed with specifications typically set for carbon‐11 labeled radiopharmaceuticals. In conclusion, this novel radiochemistry system offers a convenient GMP compliant production drugs and radioligands for imaging studies in human subjects.[ 11C‐carbonylation module allowed for GMP production of AZ13198083 in high radiochemical yield and purity.Automated  The system was in turn divided into two main subsystems. First, the radioactive gas handling system, in which [11C]carbon dioxide ([11C]CO2) was trapped from the target gas, reduced online to [11C]CO, and subsequently concentrated and transferred into the glass vial for the radiolabeling reaction. The second part was the purification and formulation subsystem, in which the crude product was first purified using high‐performance liquid chromatography (HPLC), isolated from the solvents in the mobile phase using solid phase extraction (SPE) and finally formulation in a suitable vehicle for intravenous injection and sterile filtration. The radiochemistry apparatus was controlled and monitored with Labview‐based software. A flowchart of the radiochemistry system is shown in Figure The [311C]CO and its delivery into a sealed 3.5‐ml glass reaction vessel. Thus, a series of short [11C]CO2 productions were conducted using set irradiation conditions, 35 μA for 1 min, to calculate the efficiency of each step of the radioactive gas handling system. The starting radioactivity for the process was estimated from three consecutive [11C]CO2 productions that were trapped on an ascarite trap installed at the radioactive gas inlet to the radiochemistry apparatus (Table 11C]CO2 was produced in a nitrogen‐oxygen (1%) gas target, passed through a phosphorous pentoxide trap, and trapped in a stainless steel tube (1/4″) filled with Porapak Q (80–100 mesh) that was immersed in liquid nitrogen. The trap was flushed with He, first at −196°C and then while thawing under a stream of compressed air, with the intent to purify [11C]CO2 from residual target gas and other chemical impurities. As expected, [11C]CO2 could be efficiently trapped and released with this experimental setup (Table m) than that obtained with molecular sieves, which continually traps CO2 from the atmosphere. On the other hand, a molecular sieve setup does not require any liquid nitrogen and may be more efficient in removing chemical impurities from the produced [11C]CO2. No additional traps were used to purify [11C]CO2 before it was introduced into the next step of the production sequence.The initial focus was to develop a reliable process for the production gaseous 1CCO and 11C]CO was prepared by online reduction of [11C]CO2 in a stream of helium through a heated column filled with Molybdenum powder. Though the conversion of [11C]CO2 to [11C]CO was reproducible under these conditions, it is worth noting that a pressure increase was observed following repeated use of the column, which necessitated its replacement after approximately 50 runs. It is possible that an additional purification of the [11C]CO2 could increase the lifetime of this column, but no additional efforts were put into this given the relative low replacement frequency of the Mo column. The produced [11C]CO was purified over an ascarite trap and finally concentrated from the gas stream in a stainless‐steel tube (1/16″) filled with silica (~110 mg) before being released into a sealed glass vial containing the 11C‐aminocarbonylation reaction mixture.[11C]CO to minimize the dead volume between the silica trap and the reactor. Although the pressure was not measured in the reaction vessel, it is likely governed by the vapor pressure of tetrahydrofuran (THF) at the reaction temperature during the 11C‐aminocarbonylation reaction. In the current study, in which the reaction temperature was 100°C, the pressure would thus be expected to reach approximately 2.8 bar. It is important to note that the reaction vessel is not vented until the end of the 11C‐aminocarbonylation reaction, which introduces a risk of radioactivity leakage in case the septum of the vessel leaks. However, in our experience, this risk can be efficiently managed by a simple leak check of the reaction vessel prior to its installation into the radiochemistry apparatus. We also considered introducing the [11C]CO into a vented vial to simplify the [11C]CO transfer, because a single pass introduction of [11C]CO can proceed with as high trapping efficiency as 95%.11C]CO into the reaction vessel within 7 min after end of bombardment (EOB) with a radiochemical yield (RCY) of 71% based on cyclotron produced [11C]CO2  into a prefilled vial with saline (5 ml) before it was passed through a sterile filter into a sterile product vial, also prefilled with saline (8.5 ml) to provide the end radiopharmaceutical product in a sterile solution containing less than 10% ethanol. After each synthesis, the system is automatically cleaned using a validated cleaning procedure.3.211C]AZ13198083, a radioligand for imaging the histamine type‐3 receptor . Though tests for sterility and bacterial endotoxins were outside the scope of the current study, our experience is that it is rather straightforward to manufacture sterile and endotoxin free PET radiopharmaceuticals with a suitable cleaning routine. In this context, it is important to also note that a fully automated cleaning protocol allows for the unit to be used to deliver a new batch within 2.5 h after completed production without an unnecessary radiation burden on the operator.To evaluate the efficiency of the radiochemical synthesis apparatus, a process evaluation was performed, which composed the production of 1CAZ13198r Scheme . Gratify11C‐aminocarbonylation reactions can, in our experience, be improved by dissolving all the components of the reaction mixture  in THF (~500 μl) and then purging the solvent to dryness under nitrogen gas flow (~50 ml/min) prior to adding a solution of the appropriate amine shortly before starting the radiopharmaceutical production. Though one may speculate that the oxidative addition product is formed during this process, we have no experimental support for this. Nevertheless, this “premixing” of Pd, ligand, and aryl halide was successfully adopted in the current study.The efficiency of 411C]carbon monoxide as the labeling precursor has been developed. The apparatus is setup for manufacturing according to GMP and comprises all relevant parts of a radiopharmaceutical production, including radiochemistry, purification, formulation, and sterile filtration. During process evaluation, the apparatus was successfully applied to the production of the histamine type‐3 receptor radioligand [11C]AZ13198083 in high RCY and fulfilling all set QC specifications. The development of this apparatus paves the way for future applications of [11C]carbon monoxide radiochemistry to the production of 11C‐labeled drugs and radioligands in a GMP compliant fashion for PET imaging in human subjects.A fully automated radiochemical synthesis apparatus that enables use of the versatile synthon [55.111C]CO2 production was performed using a GEMS PETtrace cyclotron . The 14N11C reaction was employed in a pressurized gas target containing nitrogen (nitrogen 6.0) and 1% oxygen (oxygen 4.8) by bombardment with a proton beam (16.5 MeV). HPLC was performed using a Hitachi L‐6200 gradient pump and a Hitachi L‐4000 variable wavelength UV‐detector in a series with a Bioscan β+‐flow detector. Analytical HPLC was performed using a Hitachi L‐6200 gradient pump and a Hitachi L‐4000 variable wavelength UV‐detector in series with a Bioscan β+‐flow detector. The reverse phase column  was eluted with a gradient between acetonitrile (A) and 100mM HCO2NH4 (B). The gradient was linear between 10% and 90% (B) over 5 min at a flow rate of 3 ml/min. Identification of all radioactive products was confirmed by co‐elution with the corresponding nonradioactive compound.Unless otherwise stated, all reagents and solvents were obtained from Sigma‐Aldrich (Sweden) and used without further purification. No‐carrier‐added [5.211C]CO2 was trapped on a Porapak Q column  immersed in liquid nitrogen. The Porapak trap was first flushed with Helium at a flowrate of 400 ml/min for 90 s while still immersed in liquid nitrogen and then flushed during thawing with heated compressed air (flowrate 50 ml/min for 40 s) before [11C]CO2 was released to the Mo column for reduction. [11C]CO2 was reduced online to [11C]CO using a preheated oven (850°C) quartz glass column (6 × 4 × 220 mm: o.d. × i.d. × length) charged with Mo powder . Unreacted [11C]CO2 was subsequently removed by a sodium hydroxide‐coated silica  trap  after which the [11C]CO was concentrated on a stainless steel trap (1/16″) filled with silica gel , immersed in liquid nitrogen. After completed entrapment, the trap was heated to release the [11C]CO through a capillary tubing  into a closed vial  prefilled with 11C‐aminocarbonylation reagents. The resulting solution was then allowed to react at the desired time and temperature. After completed reaction, the crude reaction mixture was diluted with mobile phase and transferred in two portions into an HPLC loop  using a 2.5 ml syringe pump. The solution was then injected onto the HPLC column with an injection valve  for purification. The HPLC column effluent was monitored with an ultraviolet detector (Knauer smartline UV detector 200) in series with a radiation detector . In addition, the system was equipped with a column scanner, which consists of a radiodetector moving at low speed (15 mm/s) along the HPLC column, to permit the operator to follow the movement of radioactivity inside the column during the separation. The purified product was then collected from the HPLC column into a prefilled vial with sterile water and then pushed through an SPE cartridge to collect the radiopharmaceutical product. Following a rinse with sterile water, the product was next eluted with ethanol (1.5 ml) into a prefilled vial with saline (5 ml) before it was passed through a sterile filter into a sterile product vial, also prefilled with saline (8.5 ml) to provide the end radiopharmaceutical product in a sterile solution containing less than 10% ethanol. Thirteen two‐port, two‐way valves  as well as 38 three‐port, two‐way valves  were used to direct liquid and gas flow throughout the production procedure. The entire synthesis process was controlled and monitored by an in‐house developed software (NI Labview). The movement of radioactivity in the system was monitored with a series of radiation detectors .The flowchart of the radiochemistry system is shown in Figure 5.32[π‐cinnamyl]Cl2 , xantphos , ((2S)‐2‐(4‐bromophenyl)cyclopropyl)(4‐cyclobutylpiperazin‐1‐yl)methanone (3.3 μl) were dissolved in anhydrous THF (700 μl), and the resulting mixture was flushed with N2 until the THF was fully evaporated (20 min). A solution of ethylamine in THF  was added, and the reaction vial was subjected to a leak check prior to its installation into the radiochemistry system. The 11C‐carbonylation reaction was performed during 5 min at 100°C, after which the crude product was purified using a semipreparative HPLC column  eluted with an isocratic mobile phase (30% MeCN in HCO2NH4 (0.1M)) at 6 ml/min. The eluate was monitored for absorbance (λ = 254 nm) and for radioactivity. [11C]AZ13198083 (tR = 8 min) was collected and diluted with water (~50 ml) and then concentrated on a C‐18 Sep‐Pak . The Sep‐Pak was washed with water (~8 ml) prior to elution with ethanol  into saline (5 ml). The formulation was finally filtered through a sterile filter  into a prefilled vial containing an additional portion of saline (8.5 ml) to yield the final product in a sterile injection vial and in a solution suitable for intravenous injection.Pd


A mutant (oen1) restored non‐vitreousness and produced a large cavity in the seed due to severely depleted endosperm filling. When oen1 was introgressed into inbred W64A with a normal O2 gene, the seeds appeared vitreous but had a shrunken crown. oen1 was determined to encode Shrunken1 (Sh1), a sucrose synthase . Maize contains three SUS‐encoding genes  with Sh1 contributing predominantly to the endosperm. We determined SUS activity and found a major and minor reduction in oen1 and o2, respectively. In o2;oen1-1, SUS activity was further decreased. We found all Sus gene promoters contain at least one O2 binding element that can be specifically recognized and be transactivated by O2. Sus1 and Sus2 promoters had a much stronger O2 transactivation than Sh1, consistent with their transcript reduction in o2 endosperm. Although sus1 and sus2 alone or in combination had no perceptible phenotype, either of them could dramatically enhance seed opacity and cavity in sh1, indicating that transactivation of Sus1 and Sus2 by O2 supplements SUS‐mediated endosperm filling in maize. Our findings demonstrate that O2 transcriptionally regulates the metabolic source entry for protein and starch synthesis during endosperm filling.The endosperm‐specific transcription factor Opaque2 (O2) acts as a central regulator for endosperm filling, but its functions have not been fully defined. Regular  Because many o2 mutants create an opaque mature kernel phenotype, a mild reduction in the synthesis of starch or storage proteins resulting from mutations in o2 enhancers may not cause a visible change in the phenotype of o2 seeds. A previous platform used γ‐irradiation to mutagenize a vitreous o2 variety, quality protein maize (QPM) and created many opaque QPM variants to identify o2 modifier genes  Opaque2 (O2) functions as a central player that not only transcriptionally regulates expression of most zein storage protein‐encoding genes but also directly and indirectly regulates starch synthesis  that modify the endosperm texture, creating a vitreous, hard phenotype. This mutagenesis method could identify o2 modifier genes themselves, or unrelated genes that simply exacerbate the o2 phenotype (o2 enhancers). If an o2 enhancer is mutated, endosperm modification in QPM could be disrupted and produce the opaque phenotype. Thus, we treated pollen of K0326Y, a QPM inbred line, with EMS and applied it to the associated ears. The resulting seeds were planted for self‐pollination, yielding 1500 M1 ears. One ear from this screen was found to segregate progeny kernels with normal (vitreous and plump) and mutant (opaque and shrunken) phenotypes in a 3:1 ratio . The 100‐kernel weight (KW) of oen1 in the K0326Y background (K0326Y‐oen1) was only 11.00 g, which was 36% lower than that of normal K0326Y  for two generations and then self‐pollinated for two generations. In the second backcrossing, each plant was numbered and self‐pollinated, while the pollen was used for backcrossing. Approximately one quarter of the self‐pollinated ears segregated progeny seeds with four different phenotypes, that is vitreous and plump , vitreous and shrunken , opaque and plump , and opaque and shrunken (indicative of homozygotes for o2 and oen1). Subsequently, seeds from the corresponding backcrossed ears were planted and should have produced equal amounts of four different genotypes . The WT, two single‐gene mutants and the double mutant were isolated by self‐pollinating the four different plants. The homozygous ears for each genotype resulting from one additional propagation are shown in Figure Because QPM is homozygous for o2, the oen1 and o2;oen1 double mutants had normal seed sets , thereby allowing for a little light translucency (indicated by arrows). The oen1 seeds exhibited a shrunken phenotype in the crown area, but vitreous endosperm could be formed, although it was less than WT. The double mutant seeds were dramatically shrunken and contained almost no vitreous endosperm. On a light box, the o2;oen1 seeds were even more opaque than the o2 seeds  of t Figure . A signid Figure . The conoen1 into W64A, self‐pollinated plants with the O2/O2;oen1/Oen1 genotype segregated plump and shrunken seeds at a ratio of 3 : 1 . Comparison of the single‐nucleotide polymorphism (SNP) index between the mutant and WT pools revealed a 28‐Mb region, containing nine candidate genes, near the short arm of chromosome 9  of Zm00001d045042, which could result in defective RNA splicing of this intron  to analyse the expression of Sus genes in 12‐DAP endosperms of the WT and o2. As shown in Figure Sus1 and Sus2 were dramatically reduced to a barely detectable level in o2 compared with WT. Although expression of Sh1 was downregulated in o2, the fold changes were not comparable to those for Sus1 and Sus2, consistent with our previous published RNA‐seq data  reporter gene driven by the cauliflower mosaic virus 35S promoter was used as an internal control, and the Sus gene promoters were fused with the firefly luciferase coding sequence, yielding the reporter vectors PSh1‐LUC, PSus1‐LUC and PSus2‐LUC. The effector plasmid was generated by fusing the coding region of O2 to the 35S promoter (35S‐O2). Coexpression of PSh1‐LUC, PSus1‐LUC or PSus2‐LUC with 35S‐O2 all resulted in a significant increase in LUC activity. The Sus2 promoter exhibited the strongest activation by O2 (100‐fold), followed by the Sus1 promoter (20‐fold), whereas the Sh1 promoter exhibited only a 10‐fold increase . Sh1 and Sus1 genes were found to bear one O2 box within 800‐bp promoter regions, and Sus2 had two such motifs. Oligonucleotides (50‐60 bp) labelled with FAM fluorescent molecules were used as probes to examine the binding affinity. Binding of the His‐O2 fusion protein and the His‐O2‐bZIP(aa229‐293) fusion protein to the probes could be visualized as retarded bands in the gel. The results showed that O2 and the O2‐bZIP domain protein could specifically bind to the probes from all Sus gene promoters ; each of these mutants harbour a stop codon in the coding region. Consistent with previous reports  and sus1 was created by screening their recombinants. As shown in Figure sh1 and sus1 or sus2 resulted in the seed cavity being dramatically enlarged compared with that of the sh1 single mutant. This enlargement was further enhanced in the triple mutant, which exhibited the largest hollow seed compartment among all the mutants. Similar to o2;oen1, the sh1;sus1 and sh1;sus2 double mutants and the sh1;sus1;su2 triple mutant had full seed sets on the ears. Due to severely insufficient endosperm filling, seeds in the central cob of these plants were all non‐vitreous and extremely shrunken, forming flattened kernels during seed desiccation  CWI2 and SUS function complementarily in a parallel way. Each of these genes alone is insufficient for efficient sucrose cleavage and effective endosperm filling; (ii) CWI2 and SUS work sequentially to influence endosperm development and filling. Both outcomes support the idea that the products of the two enzymes contribute to endosperm filling, but differ in location and reaction mechanisms and outcomes. The CWI2 pathway for sucrose cleavage produces twice as much hexose (fructose and glucose) as the SUS pathway (fructose and UDP‐glucose). Both mn1 and the sus triple mutant manifest dramatic reductions in endosperm size and weight, but mn1 exhibits additional developmental defects at the BETL at a relatively early stage. It is generally accepted that glucose creates signals that trigger BETL cell division and differentiation  was isolated by recurrently backcrossing to W64A (a WT inbred line) for two generations. The homozygous oen1‐1 ears were obtained by self‐pollination of BC2 seeds for two generations.The QPM line K0326Y is an 1 hybrids of oen1 and sh1‐ref. The sh1‐ref seeds were generously provided by Professor Yubin Li from the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences. To further verify the action of sh1 as an o2 enhancer, another mutant that enhances the o2 phenotype was created by EMS mutagenesis. EMS‐treated pollen of W64A was applied to the silks of W64Ao2 ears. The resulting seeds were planted for self‐pollination to screen for ears that segregated progeny with an enhanced o2 phenotype. Due to the allelism of oen1 and the new mutant, these alleles were named oen1‐1 and oen1‐2, respectively.An allelism test was carried out by gene complementation in the FA dozen mature kernels of each genotype were ground to fine powder using a tissue grinder for the analysis of the soluble sugar, starch and protein content. All measurements for each genotype were performed in at least triplicate.et al., For the measurement of soluble sugar content, 100 mg of mature kernel flour was weighed and added into 80% (vol/vol) ethanol to extract soluble sugars. The anthrone method for determining soluble sugar content was previously described  based on the activities of thermostable α‐amylase and amyloglucosidase. Each sample was measured four times.The total protein content in the mature seed flour was determined with using a Dumas rapid nitrogen determination analyser . Approximately 50 mg of seed flour was weighed into tin boats without pretreatment and pressed to pellets using a manual pressing tool. The nitrogen content was then detected by a thermal conductivity detector. Analyses were run using a standard method implemented in the instrument software, with a total analysis time of approximately 5 min. A protein factor of 6.25 was applied to calculate the average protein content. Each sample was measured three times.1F2 ears of K0326Y‐oen1 and W64A with the segregating seeds of WT and oen1 were used to clone the oen1 gene by the mapping‐by‐sequencing approach. 150 seeds of each phenotype in the BC1F2 ears, and 30 seeds of each parent, K0326Y‐oen1 and W64A, were selected and germinated. The resulting seedlings were used individually to extract genomic DNA. The individual genomic DNA for each phenotype/parent was pooled equally. Four pooled libraries with insert sizes of approximately 350–500 bp were prepared and sequenced on a HiSeq X™ Ten (Illumina) lane using 150‐base paired‐end sequencing. To identify the mutation site, the paired ‐end short reads obtained from the four samples were aligned to the reference genome of Zea mays (AGPv4.35) using BWA‐MEM software  in a 2‐mL tube at room temperature for more than 2 h. The mixture was centrifuged at 17370 g for 10 min, and 100 μL of the supernatant was transferred to a new tube and mixed with 10 μL of 10% SDS. A Concentrator Plus (Eppendorf) was used to dry the solution, and the material was then redissolved with 100 μL of ddH2O. The precipitate was extracted prior to zein buffer three additional times for non‐zein extraction. The precipitate was dried in the Concentrator Plus and vortexed with 0.5 mL of non‐zein extraction buffer  at room temperature for more than 2 h. After centrifugation at 17370 g for 10 min, the supernatant containing non‐zein proteins was transferred to a new tube. Then, 3 μL of the zein and non‐zein proteins was analysed by SDS‐PAGE (15%).Three kernels were taken from each genotype, and the embryos were removed. The endosperms were ground to a powder in a steel pot in a tissue grinder, and 50 mg of each sample was used for zein extraction with 0.5 mL of zein extraction buffer (3.75 m49EFDALFDSDKEKYAP63). Total protein in the four 12‐DAP endosperm samples was extracted as described above and separated by SDS‐PAGE (4%–20%). The proteins were electrophoretically transferred to a Bio‐Rad PVDF membrane that was activated by methanol. The membrane was processed according to the manufacturer's instructions, and the Sh1 protein was detected by the Tanon‐5200 system. The dilutions of the antibodies against Sh1 and ACTIN  were 1:1000 and 1:8000, respectively.A peptide antibody for Sh1 was prepared by Abclonal, China, in rabbits. The specific antibodies were produced using sequences unique to maize Sh1 with the following sequence: , 1% SDS and 5 mm EDTA (pH 8.0). The samples were rapidly homogenized using a tissue grinder and subsequently extracted twice using an equal volume of phenol–chloroform (pH 4.2) and an equal volume of chloroform once. The aqueous phase was transferred to TRIzol (Invitrogen) for further extraction of total RNA, followed by RNA purification using the RNeasy Mini Kit (Qiagen). A total of 5 μg of total RNA from each sample were used for reverse transcription with the Superscript III First Strand Kit (Invitrogen). RT‐qPCR was performed with SYBR Green (Takara) on a Bio‐Rad CFX‐96 thermocycler. The relative expression of these genes was calculated using the ΔΔCt method. The internal control was the maize Ubiquitin gene. All primers used in this experiment are listed in Table All materials for RNA extraction were kept at −80 °C until use. For total RNA extraction of developing seeds and endosperms, samples were placed in a 2.0‐mL microfuge tube with 200 μL of RNA extraction buffer containing 150 mEndosperms separated from fresh developing seeds were rapidly frozen in liquid nitrogen and kept at −80 °C until use. More than 20 endosperms were immersed in a steel tank cooled with liquid nitrogen and rapidly ground to a powder in a tissue grinder. 50 mg of endosperm powder was immediately placed into a tube cooled with liquid nitrogen to measure SUS activity. The Solarbio Sucrose Synthetase (SS) Assay Kit  was used for activity determination. Sucrose synthetase catalyses the conversion of fructose and UDPG to UDP and sucrose. The reaction between sucrose and resorcin results in a colour change, with a characteristic absorption peak at 480 nm, and the enzyme activity is proportional to the colour intensity. Each sample was measured six times. We used ultraviolet visible photometer (Beckman Coulter DU730) to detect the characteristic absorption peak of 480 nm. Each sample was measured six parallel experiments.2O with shaking incubation for 30 min on ice. The supernatant was then moved to a new centrifuge tube after centrifugation at 13 000 rpm for 10 min at 4 °C and filtered with 0.22 μm filter. The filtered liquid was diluted with ddH2O by 10 fold for measurement by the ion chromatography  using a CarboPac PA‐20 column  and solvent B (200 mm NaOH).Fifty mg of freshly endosperm prepared powder was extracted with 2 mL ddHet al., O2 driven by the 35S promoter. The reporter vector pGreenII 0800‐LUC was used to detect the transactivation of the three Sus gene promoters. The LUC/REN activity ratio was measured using the DLR assay system (Promega). Primers are listed in Table Isolation of Arabidopsis mesophyll protoplasts, PEG–calcium transfection of plasmid DNA, and protoplast cultivation were performed as described previously (Zhang 229‐293) and the EMSA procedure have been described previously  was used to calculate The authors have no conflict of interest to declare.YW conceived the idea and supervised the project overall. YD and YW wrote and revised the manuscript together. YD designed and performed the experiments. JW and ZZ participated in the experimental design, phenotype measurements and manuscript revision.Figure S1 The K0326Y‐oen1 mutant generated by EMS‐induced mutagenesis of K0326Y.Figure S2 The plant phenotype of WT, o2, oen1‐1, o2;oen1‐1 in the W64A background.Figure S3 SDS‐PAGE analysis of zein (upper) and non‐zein (lower) proteins of mature kernels of WT, o2, oen1‐1, o2;oen1‐1 in the W64A backcross population.Figure S4 The phenotype of oen1‐1 in the W64A background.Figure S5 Genetic complementation test of oen1‐1 with sh1‐ref and oen1‐2 alleles.Figure S6 Kernel phenotypes of WT, o2, oen1‐2, o2;oen1‐2 in the W64A background.Figure S7 SDS‐PAGE analysis of zein (upper) and non‐zein (lower) proteins of mature seeds of WT, o2, oen1‐2, o2;oen1‐2 in the W64A background.Figure S8 SUS activity in developing endosperms of WT, o2, oen1‐1, o2;oen1‐1 in the W64A background at 10 and 14 DAP.Figure S9 Levels of sucrose (a), glucose (b) and fructose (c) in developing endosperms of WT, o2, oen1‐1, o2;oen1‐1 in the W64A background from 8 to 24 DAP.Figure S10 Alignment analysis of amino acid sequences of the three SUS proteins.Figure S11 Kernel phenotypes of WT and single, double and triple mutants of the three Sus genes.Figure S12 Levels of sucrose (a), glucose (b) and fructose (c) in 12DAP endosperms of WT and single, double and triple mutants of the three sus genes.Table S1 Primers used in this study.Appendix S1 The promoter information of Sh1, Sus1 and Sus2.Click here for additional data file. Click here for additional data file.Supplemental Dataset File Original data and details for all statistical analyses in this study.Click here for additional data file. Click here for additional data file.


Heterocycles, a class of molecules that includes oxazoles, constitute one of the most common building blocks in current pharmaceuticals and are common in medicinally important natural products. The antitumor natural product nataxazole is a model for a large class of benzoxazole‐containing molecules that are made by a pathway that is not characterized. We report structural, biochemical, and chemical evidence that benzoxazole biosynthesis proceeds through an ester generated by an ATP‐dependent adenylating enzyme. The ester rearranges via a tetrahedral hemiorthoamide to yield an amide, which is a shunt product and not, as previously thought, an intermediate in the pathway. A second zinc‐dependent enzyme catalyzes the formation of hemiorthoamide from the ester but, by shuttling protons, the enzyme eliminates water, a reverse hydrolysis reaction, to yield the benzoxazole and avoids the amide. These insights have allowed us to harness the pathway to synthesize a series of novel halogenated benzoxazoles. Benzoxazole biosynthesis, a key step in the biosynthesis of the antitumor agent nataxazole, proceeds via a hemiorthoamide intermediate that arises from an unstable ester. NatL2 catalyzes the formation of this ester that spontaneously rearranges into an off‐pathway amide. In the presence of NatAM, the ester is converted to benzoxazole via a hemiorthoamide intermediate. These enzymes are used to synthesize a series of novel halogenated benzoxazoles. Over 80 % of currently prescribed small molecule drugs contain a heterocyclic ring.Unconjugated oxazoles are mostly produced by ribosomally synthesized post‐translationally modified peptides (RiPPs) synthesis and by non‐ribosomal peptides (NRPs) synthesis.Streptomyces chartreusis NRRL 3882), nataxazole (S. sp. Tü 6176), A33853 (S. sp. NRRL12068), and caboxamycin (S. sp. NTK 937), have been identified , catalyze the formation of CoA thioester derivatives by two half reactions: adenylation of the carboxylic acid, and subsequent thioesterification. Non‐thioesterifying acyl‐CoA ligases, are known but rare, found only in fatty acyl‐AMP ligases (FAAL)We have determined the structures of both NatL2 (homologue of BomJ) and NatAM (homologue of BomN) from the antitumor nataxazole Figure  d pathwaE. coli and S. albus, respectively (NatAM expressed in E. coli was insoluble). HPLC analysis detected the benzoxazole, benzoxazole‐4‐carboxylic acid) in the presence of NatL2, NatAM, ATP, 3‐HAA, and MgCl2 , 1H‐NMR and 13C‐NMR spectroscopy  confirmed 3. When NatAM, NatL2, ATP or 3‐HAA were individually omitted, 3 was not detected.NatL2 and NatAM were expressed and purified as N‐terminal hexahistidine‐tagged fusion proteins in 2 Figure  f, in ag2, (2‐amino‐3‐((2‐carboxy‐6‐hydroxyphenyl)carbamoyl)benzoic acid), was detected  following incubation of NatL2 with ATP, 3‐HAA, and MgCl2. We incubated 2 with NatAM but no 3 or consumption of 2 were detected  indicating a very close molecular relationship. Upon standing, 1 converts to the amide 2 , whilst NatAM addition yields the product 3  leading to the conclusion the Zn2+ plays an essential structural role.We determined the 1.9 Å‐resolution crystal structure of NatL2 in the presence of ATP ). The Zn2+ is a unique feature of NatL2. The adenine and ribose rings of AMP superimpose in the structures; the α‐phosphate is displaced by 1.2 Å. In NatL2, the adenine ring is sandwiched between the main chain peptide bonds of Ala212 to Pro214 on one side and Tyr236 on the other. In broad terms, the binding site of AMP in NatL2 is conserved in PaaK. However, in NatL2, the α‐phosphate makes a salt bridge with Lys418 from the other monomer , whilst in PaaK (PDB 2Y27) Lys422 in the same monomer plays this role. The salt bridge between lysine and the α‐phosphate is a feature of these enzymes,NatL2 resembles acetyl‐CoA synthetases, with an rmsd of 1.9 Å over 362 Cα atoms with PaaK from 2+. We soaked the AMP crystals with both AMPPNP and 3‐HAA overnight and saw clear density for both molecules  consistent with adenylation of the carboxylate. Thr239 hydrogen bonds to both α‐phosphate and carboxylate oxygen atoms. The hydroxyl group of 3‐HAA is hydrogen bonded to Glu235 whilst the amino group makes a hydrogen bond to N7 of the adenosine ring. The benzene ring stacks against a stretch of the main chain of Gly237 and Ser238. This complex is model for the Michealis complex of the first half (adenylation) reaction  . The β‐phosphate of AMPPNP interacts with the side chains of Ser87, Ser88, and Thr239, and backbone amides of Ser88 and Thr239, while the γ‐phosphate has fewer interactions making hydrogen bonds with a water molecule and the side chain of Ser88. There was no electron density for bound Mg1) binds almost identically to the molecule in the AMPPNP:3‐HAA complex. There is residual electron density suggesting that we could fit a second molecule of 3‐HAA (3‐HAA2) bound adjacent to the 3‐HAA1 molecule; the orientation suggested that the hydroxyl not the amine would attack the 3‐HAA1‐adenylate . We reasoned the low quality of the residual density arose from the crowding of carboxylate of 3‐HAA1, hydroxyl of 3‐HAA2 and the α‐phosphate of AMP. We therefore soaked the co‐crystallized AMP:3HAA crystals with salicylic acid (SA), which lacks the hydroxyl at the meta position. In subunit A, two molecules of SA are bound but no AMP  of 3‐HAA2 upon the 3‐HAA1‐adenylate to yield the ester . 3‐HAA oxy)benzoic acid  and 3‐oxy)‐2‐hydroxybenzoic acid (5) . SA, which lacks a hydroxyl group at the meta position, gives no reaction, indicating that the hydroxyl must be at the meta position for reaction. The formation of esters with substrate analogues with a meta hydroxyl is evidence that NatL2 catalyzes ester formation with 3‐HAA. We assign compound 1 8 arrangement where it is coordinated by His75, His77, His253, and one water molecule in an approximate tetrahedral arrangement .The structures of NatAM with 3‐HAA (1.25 Å resolution) and 3‐HBA (1.17 Å resolution) were obtained by soaking and show that 3‐HAA and 3‐HBA molecules superimpose , as do the protein structures, which are largely unchanged from the 4  has adjusted its conformation  so that Arg87 salt bridges to the carboxylate of the other ring  of the ester  of 74°, consistent with attack to form a hemiorthoamide intermediate . A water molecule bound to Lys315 and the ester is conserved in the structures. The mutant K315A shows a 10‐fold reduction in activity .We soaked NatAM crystals with a substrate mimic, ester 12 (the fluorine atoms may deactivate the carboxylate for adenylation). The system was able to process analogues with chloro and bromo substituents at C5. These halogens would either require significant movement of the Tyr227 in NatAM or alternatively that the phenyl ring rotates by 180° around its C1–C4 axis such that that the bromo and hydroxyl substituents swap binding sites. That the system tolerates a wide range of substrates shows it holds promise as a synthetic tool.Halogenation is a very common medicinal chemistry modification, fluorine especially has a unique chemistry.2+. NatL2 belongs to the PaaK family, a subset of acyl‐CoA synthetases that operate via acyl adenylates. To enable CoA binding and thus thiol attack on the acyl‐AMP intermediate, PaaK enzymes undergo a major conformational change where the C‐terminal domain rotates to make an “open” form. This motion is prevented in NatL2 by a C‐terminal extension that locks the structure in the “closed” form . FAALs are locked in the “closed” arrangement and unable to catalyze CoA thioester formation due to an insertion into the hinge between the C‐ and N‐terminal domain.The biosynthesis of benzoxazoles in contrast to (thi)oxazole biosynthesis has remained uncharacterized, an important oversight given its prevalence in medicines and natural products. We reconstituted the biosynthesis of the benzoxazole moiety found in nataxazole by adding two enzymes , to 3‐HAA, ATP, and Mg2 is not the true product of NatL2, rather NatL2 catalyzes an ester linkage between two 3‐HAA molecules, compound 1. We conclude that the previous report2 to cells with a deletion mutant (ΔbomO) in the 3‐HAA pathway was previously shown to result in a small amount of benzoxazole.2 to 3‐HAA that is then processed to the product.Previous studies had predicted that BomJ, (NatL2 homologue) synthesized an amide linkage between two 3‐HAA monomers via an acyl‐AMP intermediate.1 spontaneously converts to 2 so rapidly it prevented isolation of 1 in our hands explaining the isolation of 2 previously.1 is predicted to occur via a tetrahedral hemiorthoamide intermediate formed by attack of the amine on the ester  should be addressed to the authors.SupplementaryClick here for additional data file.


A similar modified nucleoside, 2‐thiocytidine, was recently found to be sulfur‐methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4‐methylthiouridine (ms4U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms4U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable‐isotope pulse chase studies (NAIL‐MS) and showed its AlkB‐independent removal in vivo. Our findings reveal the complex nature of bacterial RNA damage repair.Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4‐thiouridine (s Through a combination of synthetic and analytical chemistry, the structure of the modified nucleoside 4‐methylthiouridine was confirmed in bacterial tRNA. The modification is of low abundance and forms after bacterial stress induced by methylating agents; this argues for its nature as a natural tRNA lesion. By pulse chase analysis, we show its repair through a dethiomethylation mechanism in vivo. Similar to SLAM‐Seq, TUC‐Seq uses metabolically introduced s4U, which can be chemically converted to cytidine prior to RNA sequencingRNA and especially tRNA have complex structures to fulfill their important functions inside the organism. This is possible through the vast chemical variety of building blocks found in RNA. To date over 170 modifications to either ribose or nucleobase have been reported.2C)  or antibiotics (streptozotocin) was observed. The resulting damage ms2C .To address these questions, we report here the synthesis of the suggested damage product ms4U (4) was first attempted via the formation of the fully acetylated corresponding 4‐triazolic precursor which was meant to react with sodium thiomethanolate2) separately with subsequent methylation adopting a procedure for the corresponding 2′‐chlorine riboside.1) in high yields. The subsequent formation of the 4‐thiouridinic compound (2) by thiolation with phosphorus pentasulfide yielded 72 %. It should be noted, that crystallization from ethanol, as described for the chlorinated compound, could not be observed. The deprotection was conducted by refluxing in concentrated aqueous ammonia solution, and s4U (3) was received presumably in quantitative yield but was used as crude product in the next step. Of note, the more common method under Zemplén conditions2). In a final step, the thio group was selectively methylated by iodomethane to provide ms4U (4) in a moderate overall yield of 40 % over four steps.The synthesis of ms4U in tRNA from unstressed Escherichia coli. With this targeted analysis, we found a peak in native tRNA that corresponds to the synthetic ms4U in terms of retention time, precursor and product ion mass. In E. coli exposed to the LD50 dose of methyl methanesulfonate (MMS), the peak increased. A co‐injection of the synthesized ms4U standard and tRNA from MMS exposed E. coli grown in stable isotope labeled medium clearly showed 1) perfect co‐elution and 2) the expected numbers of carbon, nitrogen, and sulfur atoms in native ms4U  of ms4U was produced by metabolic isotope labeling of E. coli. To increase the yield of stable isotope labeled ms4U, MMS was added to the culture medium for 60 minutes, and the RNA was harvested and processed as previously described.4U and metabolically produced ms4U‐SILIS allowed accurate quantification of ms4U and other modified ribonucleosides in bacterial tRNA .We were next interested to quantify the extent of msA Figure . For norE. coli, we found 2.6×10−6 ms4U/rN, which is less compared to the natural abundance of our recently described modification ms2C (17×10−6 ms2C/rN).In tRNA of unstressed −6 m1A/rN, 1543×10−6 ms2C/rN, 2772×10−6 m7G/rN, 530×10−6 m3C/rN, 478×10−6 m6A/rN and 41×10−6 m3U/rN  a different conclusion must be drawn. With 0.5 % ms4U/s4U, ms4U is of comparable abundance to the known damage product m1A , for example, m/z (s4U) 261. In this medium, the bacteria are exposed to MMS (20 mM) and s4U is converted to ms4U and, for example, A to m1A. After exposure, MMS is removed by exchanging the medium with stable isotopes containing medium. During the following recovery period, newly transcribed tRNA will be 15N labeled, enzymatically methylated nucleosides will be CD3 labeled and new s4U will have a 34S label . The experimental design is shown in Figure 4U during MMS exposure with around 50×10−6 ms4U/original rN. In the subsequent recovery period, we traced the abundance of ms4U and normalized it to the abundance of original rN. In wild‐type E. coli, we saw a constant decrease in ms4U over time . Dethiomethylation was observed after incubation with dithiothreitol (DTT). No dethiomethylation was observed under acidic/alkaline conditions, in growth medium or in the presence of cysteine or bovine serum albumin (BSA as an example protein).While we cannot exclude the involvement of an unknown dethiomethylase, we tested the possibility of spontaneous ms4U in bacterial tRNA. The low abundance of ms4U indicates its formation as a lesion through the constantly present electrophile S‐adenosylmethionine. During the exposure of bacteria to methylating agents such as MMS, RNA is damaged, and the methylation products of canonical nucleosides  emerge.In summary, we describe the existence of thiomethylated s2C and s4U, become methylated. As evident from Figure 2C is more prone to direct methylation than s4U. This can be explained by both the chemical reactivity of the S2 in cytidine compared to the S4 in uridine and its location within the tRNA. Due to the exocyclic amine in cytidine, s2C has an increased electron density, which improves its nucleophilic character over the S4 in uridine. Furthermore, the uridine S4 is more prone to solvation, which further decreases its nucleophilicity. In addition to the difference in nucleophilicity, s2C is exposed and accessible in the anticodon loop of the tRNA, whereas s4U is buried in the D‐/T‐loop fold.In addition, modified nucleosides with a pronounced nucleophilic character, such as s1A, m3C  and ms2C . The third class of RNA damage comprises m7G, which is not removed from tRNA .Our studies reveal a differential reaction of the cells towards these forms of RNA damage. One class of lesions is repaired through enzymatic demethylation using an oxidative demethylation mechanism. Namely, mC Figure C are sub4U as a natural and stress‐induced lesion in bacterial tRNA confirms the importance of tRNA modifications during stress response.Overall, the finding of msThe 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.


Based on first‐principles calculations, it is found that in the equilibrium state, ML Na2O is a 2D double Weyl semimetal, while ML K2O is a 2D pseudospin‐1 metal. These exotic topological states exhibit a range of fascinating effects, including universal optical absorbance, super Klein tunneling, and super collimation effect. By introducing biaxial or uniaxial strain, a series of quantum phase transitions between 2D double Weyl semimetal, 2D Dirac semimetal, 2D pseudospin‐1 metal, and semiconductor phases can be realized. The results suggest monolayer dialkali‐metal monoxides as a promising platform to explore fascinating physical phenomena associated with novel 2D emergent fermions.2D materials with nontrivial energy bands are highly desirable for exploring various topological phases of matter, as low dimensionality opens unprecedented opportunities for manipulating the quantum states. Here, it is reported that monolayer (ML) dialkali‐metal monoxides, in the well‐known 2H‐MoS 2 type metal‐shrouded oxides named dialkali‐metal monoxides are predicted to host multiple topological phases with exotic quasiparticle excitations, including double Weyl, 2D Dirac, 2D pseudospin‐1 metal, and semiconductor phases. These phases can be effectively tuned and manipulated by strain engineering and can be manifested by various fascinating effects such as super Klein tunneling and super collimation effect.2H‐MoS It is motivated by recognizing that many unusual properties of graphene are connected to its special topological band structure: the conduction and valence bands linearly cross at symmetry‐protected nodal points, such that the low‐energy quasiparticles behave like Dirac fermions with zero effective mass. Currently, a target in this direction is to explore new types of band degeneracy points and the associated new emergent fermions. For example, previous works have theoretically proposed 2D massive Dirac fermions,2C, Ti2N, and Nb2C),M–X–M, where M is the transition metal and X is C or N. Recently, the study has been extended to metal‐shrouded oxides.2O with the same structure has been proposed. Considering that Tl has properties similar to alkali metal elements, it is natural to ask whether alkali metal oxides  can also be stabilized in a 2D crystal form? If so, this may open the door to a new family of 2D materials, i.e., 2D metal‐shrouded oxides. In view of the great variety of 2D metal‐shrouded MXenes and their rich properties, one can expect that 2D metal‐shrouded oxides will also grow into a big family with fascinating properties.Meanwhile, among the 2D materials family, the metal‐shrouded MXenes have been attracting great interest due to their rich properties.2 structure. Based on first‐principles calculations, we show that Na2O and K2O can be stabilized in this 2D lattice as shown in Figure2O is a 2D double Weyl (massive Dirac) semimetal, while ML K2O is a nearly ideal 2D pseudospin‐1 metal. To our best knowledge, they represent the first examples that host the 2D double Weyl semimetal and the pseudospin‐1 metal in the equilibrium state. Furthermore, the topological phase and the novel fermions can be effectively controlled by strain. Under biaxial strain, a series of topological phase transitions between 2D Dirac metal, double Weyl semimetal, pseudospin‐1 metal, and semiconductor phases can be realized. Particularly, when uniaxial strain is applied, an intriguing transition from double Weyl point to two single Weyl (Dirac) points is triggered, which would be manifested in quantum transport measurements by a drastic changeover from weak localization to weak antilocalization. This kind of phase transition has not been reported for realistic materials before. In addition, these emergent fermions will lead to a range of exotic effects, including universal optical absorbance, super Klein tunneling, and super collimation effect, which have been predicted but not experimentally demonstrated yet due to the lack of suitable materials. Thus, our discovery not only predicts a family of new 2D materials but also provides an experimentally feasible platform to explore new emergent fermions and their fascinating fundamental physical effects.In this work, we try to address the above challenge and question by proposing a new family of 2D material—monolayer dialkali‐metal monoxides (DMMOs) with the 2H‐MoS22O  take the same structure as ML 2H‐MoS2, in which three atomic layers are stacked in the A–O–A sequence with the D3h point group symmetry, forming the P6¯m2 space group (No. 187). As depicted in Figure 2O and K2O are 3.49 and 3.86 Å, respectively.The proposed ML dialkali‐metal monoxides Aroup No. 87. As de2O is shown in Figure 2O is similar to Na2O, as shown in the Supporting Information. Excellent energetic stability has also been validated, as evident by the large cohesive energy of −8.02 and −7.36 eV per formula (f.u.−1) for Na2O and K2O, respectively. These results suggest the experimental feasibility to synthesize the proposed monolayer materials. For the other DMMOs , we find that Li2O has some imaginary phonon modes while Rb2O, and Cs2O is extremely unstable. The calculation results (including their band structures) are summarized in the Supporting Information.We calculated the phonon spectra of DMMOs to investigate the dynamical stability of these compounds. The result for ML NaFigure2O and K2O. The band structures of Li2O are similar to K2O, as summarized in Figure S8 in the Supporting Information. It is noteworthy that, due to the small atomic numbers, spin–orbit coupling (SOC) has negligible effect on the band structure of DMMOs. Consequently, electron spin can be regarded as a dummy degree of freedom and the DMMOs can be treated as effective spinless systems. In the following, unless explicitly stated, we will not take spin degeneracy into account when analyzing the band topology. As shown in Figure E′ and A1′ irreducible representations of the D3h point group . The difference is mainly due to stronger ionic bonding in ML K2O. Consequently, in the vicinity of triply degenerate point, the upper and lower bands are characterized by graphene‐like linear dispersion, intersected by a very flat band at the Fermi point  band structures. Exactly at Γ point (k = 0), one can see that M1 and M2 represent the energies of the A1′ and the E′ states, respectively. By determining the band parameters of this general Hamiltonian, we can quantitatively understand the pronounced changes in the low‐energy band structures of Na2O and K2O.The triple band degeneracy point and the associated pseudospin‐1 fermions can be well modelled by the 2O, E′ has higher energy than A1′, meaning M1 is smaller than M2. With a unitary transformation, we can project the H0 (k) model onto the two E′ bands around the double Weyl (massive Dirac) point. The resulting two‐band model is written byB± = 0.5(B2 ± B1), k± = xk ± yik, and the energy is measured from the degenerate point. Without the diagonal term, the model HDW resembles that of the AB‐stacked bilayer graphene, which features a Berry phase of 2π, doubling that of a single Dirac point as in monolayer graphene.2O, there is only one double Weyl point sitting at the BZ center. The double Weyl point endows ML Na2O with an interesting optical property, i.e., it should exhibit a universal optical absorbance of πα ≈ 2.3% (α is fine‐structure constant) at low frequencies [for ℏω < (M2 – M1) here].First, for Na2O, the triply degenerate point can be explained by setting M2 = M1 = 0 (by neglecting the insignificant gap). In this case, the k‐linear terms dominate the low energy spectrum, which allows us to further simplify the Hamiltonian H0 into the following formS is pseudospin‐1 angular momentum vector.HPS1 mimics the Hamiltonian of 3D Weyl, each S matrix is 3 × 3, representing the triply degeneracy of pseudospin‐1 fermions in K2O near the Fermi surface. In contrast to blue phosphorene oxide and Mg2C, the middle intersecting band is extremely flat, nearly extending over the whole Brillouin zone. Intriguingly, ML K2O exhibits pseudospin‐1 phase in the equilibrium state, which is critical for experimental exploration of exotic quantum effects associated with pseudospin‐1 fermions, such as super Klein tunneling with almost 100% transmission probability for large incident angles,2O may provide a promising platform for observing these fascinating phenomena.For KC =1S0 ∂2Es∂ε2, where S0 represents the area of the unstrained cell and ES is the energy difference between unstrained and strained systems. Our calculation shows that the stiffness constants for Na2O and K2O are about 57.4 and 38.9 N m−1, respectively. These values are significantly smaller than archetypal 2D systems of graphene (≈340 N m−1)2 (≈180 N m−1),−1.It is well known that the physical properties of 2D materials can be effectively tuned by strain, like the bandgap engineering of 2H‐TMDC semiconductors.2O, multiple quantum phases can be generated, as shown in Figure2O becomes metallic with six extra Dirac points  is destroyed. Instead, a new pair of linearly dispersed Dirac points, as required by time reversal symmetry, emerge along Γ‐M  point at Γ is splitting into two single Dirac points located at kD±= on the xk axis . Expanding the model at kD± leads to 2D Dirac modelq is measured from kD±, ± corresponds to the two Dirac points, and v±=2B± D/B−. As we already mentioned above, the double Weyl point is characterized by a 2π Berry phase, whereas a single Dirac point corresponds to a π geometric phase. At low‐temperature, a 2π Berry phase is manifested by weak localization in quantum transport, whereas a π Berry phase leads to weak antilocalization. Thus, the uniaxial strain can induce an interesting transition from weak localization to weak antilocalization in ML DMMOs, which would be detectable by charge transport measurements. Due to the reduced symmetry, the aforementioned Dirac model also has a finite energy tilt term of ±v+xσq0, which may induce interesting squeezing effects on the Laudau level spectrum in magnetic field.2 index is one, indicating a nontrivial 2D topological insulator (TI) state. The resulting topological edge state is plotted in Figure S5 in the Supporting Information, which also shows the evolution of Wannier charge center by means of WannierTools.2O ML, making ML DMMOs promising 2D TI candidates for studying quantum spin Hall states.Next, we consider the effects of uniaxial strain, which has been experimentally employed in modifying the physical properties of graphene and TMDCs., namelyH k=HDW k2O could grow during the oxidation of Na (110) surface2O may also be exfoliated from bulk.2O is ≈0.45 eV higher per unit in energy than 1T‐K2O (≈0.65 eV for Na2O). However, these energy differences are much smaller than the energy difference between 1T and 2H phases of ML MoS2 (≈0.85 eV). Since the metastable 1T phase of ML MoS2  or by chemical vapor deposition method. Another possible method is using alkali thin substrate with adsorption of oxygen, similar to the surface selenization to get reversible transition between 2H and 1T PtSe2.It is well known that TMDCs exhibits 1T or 2H phase under different environment. Similarly, ML DMMOs also have 1T phase counterparts which are dynamically stable.32O is a 2D double Weyl semimetal, and K2O is a 2D pseudospin‐1 metal. They may host a range of fascinating physical effects such as universal optical absorbance, super Klein tunneling, and super collimation effect. Furthermore, rich topological phase transitions can be achieved in these materials by strain. Under biaxial strain, a series of transitions can be realized. Under uniaxial strain, the double Weyl point can be split into a pair of single Weyl (Dirac) points, accompanied with the transition from weak localization to weak antilocalization in quantum transport property. In addition, this phase is a 2D topological insulator state when SOC is included. Our work provides a new 2D material platform to explore new topological emergent fermions and their interesting effects, which is of significant fundamental importance. Besides, the exotic physical properties and effects associated with these fermions are expected to make the materials promising candidates for electronic device applications. The strain‐induced metal–insulator quantum phase transition also makes these materials promising for making sensitive mechanical sensors.In conclusion, we propose a family of new 2D materials—the monolayer dialkali‐metal monoxides with the 2H‐TMDC structure. These materials enjoy good stability and excellent flexibility. Most importantly, we find that these materials are novel types of 2D topological metals. In the equilibrium state, Na4−1 and 10−6 eV, respectively. Energy cutoff was set to be 500 eV and the Brillouin zone was sampled using a 23 × 23 × 1 Γ‐centered k‐point mesh. Phonon spectra were calculated based on a 4 × 4 × 1 supercell by the finite displacement method using the PHONOPY package.2O and K2O, which are the focus of the work, the effect of SOC is negligible, and hence the results without SOC are used for the discussion unless specified (see the Supporting Information).The first‐principles computations were based on the DFT, performed by implementing the projector‐augmented waveThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.