This article aims to detect the effect of SAM domain, SH3 domain, and nuclear localization signal 1 (SAMSN1) in neonatal rats with neurological dysfunction induced by hypoxia and ischemia (HI). The HI model was created using 7‐day postnatal rats. Zea‐longa score was utilized to validate the neurological injury after HI. Then, the differentially expressed genes (DEGs) were detected by gene sequencing and bioinformatics analysis methods. The oxygen and glucose deprivation (OGD) models were established in the SY5Y cells and fetal human cortical neurons. In addition, SAMSN1‐small interfering RNA, methyl thiazolyl tetrazolium assay, and cell growth curve were employed to evaluate the cell viability variation. Obviously, Zea‐longa scores increased in rats with HI insult. Subsequently, SAMSN1 was screened out, and it was found that SAMSN1 was strikingly upregulated in SY5Y cells and fetal neurons post‐OGD. Interestingly, we found that SAMSN1 silencing could markedly enhance cell viability and cell growth after OGD. These data suggested that downregulation of SAMSN1 may exert a neuroprotective effect on damaged neurons after HI by improving cell viability and cell survival, which provides a potential theoretical basis for clinical trials in the future to treat neonatal hypoxic–ischemic encephalopathy. The purpose of this study was to investigate the effects of the SAM domain, SH3 domain, and nuclear localization signal 1 (SAMSN1) in neonatal rats with neurological dysfunction induced by hypoxia and ischemia (HI). Differentially expressed genes in the HI model established by Sprague–Dawley rats were detected by gene sequencing and bioinformatics analysis, and HIE‐related SAMSN1 was screened out. SY5Y cells and fetal human cortical neurons were used to establish a cell model of oxygen and glucose deprivation‐induced injury, and the cell viability variation is evaluated by SAMSN1‐small interfering RNA, methyl thiazolyl tetrazolium assay, and cell growth curve. The results suggest that downregulation of SAMSN1 may play a neuroprotective role in HI‐impaired neurons by increasing cell survival rate and cell survival rate. NHIE is one of the main causes of neonatal death because it has a great negative impact on the brain development of newborns, and has the highest mortality and morbidity among infants worldwide. The incidence of HIE in the United States is 2–3 in 1000 fetuses, and 20%–25% of term newborns die in the neonatal period.Although HIE has complex pathophysiological characteristics, the various steps leading to cell damage provide many opportunities for therapeutic intervention. SAM domain, SH3 domain, and nuclear localization signal 1 (SAMSN1), also known as HACS1, NASH1, or SLY2, is a member of a new family of putative adaptor proteins and scaffold proteins containing SH3 and sterile alpha motif (SAM) domains,This study validated neurological damage in neonatal rats after HI using the Zea‐longa scores test. Then, SAMSN1 was screened out, and its function in developing NHIE was analyzed by bioinformatics analysis. Additionally, the effects of SAMSN1 on SY5Y cells and fetal human cortical neurons were further investigated with SAMSN1‐small interfering RNA (siRNA), 3‐‐2,5‐diphenyltetrazolium bromide (MTT) assay, and cell growth curve following oxygen and glucose deprivation (OGD). For the first time, the present study reported the close connection between SAMSN1 expression and HIE and provided a foundation for the molecular pathogenesis of NHIE in clinical trials.22.1n = 10/group).A total of 20 SD rats  were purchased from the Animal Center of Kunming Medical University and fed following the guideline of the China Laboratory Animal Protection and Ethics Committee. The study was approved by the Ethics Committee of Kunming Medical University and the Animal Care & Welfare Committee of Kunming Medical University (no. KMMU2020001) and followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The rats were kept in 45%–50% humidity, 12‐h light/dark alternating mode, the room temperature was maintained at 21–25°C, and they ate and drank freely. Neonatal rats were randomly divided into the sham and HI groups  and the HI group (n = 10) were anesthetized with 3% isoflurane (RWD). The heart was perfused with normal saline until the liver turned white. An incision was made in the skin and skull at the top of the brain, the brain was exposed, and the brain was removed and placed in 0.1 M precooled phosphate‐buffered saline (PBS). After the pia mater and blood vessels were entirely removed, the right cerebral cortex tissues were carefully removed. Total RNA was extracted from the cortical tissue surrounding the cerebral infarction site. The cerebral tissues were collected according to the manual and the gene sequencing was carried out by Biomarker Technologies .At 24 h after HI modeling, the neonatal rats in the sham group (2.5https://string-db.org/cgi/input.pl). GO analysis was employed to annotate biological processes, molecular function, and cellular components.The differentially expressed genes (DEGs) were analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis, and STRING online tools , Ham's F12 medium, and 10% supplemental fetal bovine serum (FBS) at 37°C with 5% CO2. Cells were seeded (0.5 × 104 cells/cm2), and then cultured at 37°C with 5% CO2. SY5Y cells were passaged after 2 days in culture and then used for the establishment of the OGD model.SY5Y cell lines (GeneCopoeia Company) were cultivated in flasks with a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) , the obtained suspension was centrifuged (1000 rpm) for 10 min. The pellets at the bottom were subsequently resuspended in the complete culture medium (Hyclone) containing DMEM/high glucose (Hyclone), 10% FBS, and 1% penicillin–streptomycin solution (Hyclone). Viable neurons were counted by Trypan blue exclusion and then seeded into 96‐well plates (Corning) with substrate pretreatment at 5 × 105 cells/ml, followed by incubation at 37°C with 5% CO2. The medium was replaced with a neurobasal medium with 2% B27 (Invitrogen) after 4 h and was changed the next day. Half of the medium was changed every 3 days.The cortical brain tissues were harvested from a naturally aborted fetus (29 days old) provided by the First Affiliated Hospital of Kunming Medical University, which was approved by the Ethics Committee of Kunming Medical University on September 30, 2015 . After being cut into 1 mm2.82 and 5% CO2 at 37°C for 2 h. Subsequently, the cells were re‐placed into a normal medium containing 95% air and 5% CO2 for reoxygenation for 24 h.SY5Y and fetal human cortical neurons were washed with PBS and then placed into the glucose‐free medium (Gibco) at 37°C. Afterward, the cells were placed in a chamber filled with 95% N2.92−ΔΔCt method.Total RNA was extracted from cells using Trizol reagent (Takara Bio Inc.) according to the manufacturer's protocol strictly and then reversely transcribed to complementary DNA (cDNA) with the Revert AidTM First Strand cDNA Synthesis Kit (Thermo Fisher). Primer sequences were designed by Primer 5.0 software for RT‐qPCR as follows: SAMSN1: forward, 5′‐CACTGCTGTCCGTTCTCC‐3′; reverse, 5′‐GCTCTTCCTTCTTACCCTCA‐3′; GAPDH: forward, 5′‐TGACTTCAACAGCGACACCCA‐3′; reverse, 5′‐CACCCTGTTGCTGTAGCCAAA‐3′. Next, the reaction was completed in a DNA thermal cycler , the process was as follows: one cycle of 95°C for 2 min; 40 cycles of 95°C for 15 s, annealing for the 20 s, and 60°C for 40 s. Data were counted through the normalization of GAPDH values through the 2.10Gene database of SAMSN1 from the National Center for Biotechnology Information was firstly obtained, and then three siRNAs inhibiting SAMSN1 expression and one noninhibitory and Cy3‐labeled siRNA as negative control (NC) were designed and acquired from Ribobio Company. Briefly, when the fusion rate of SY5Y cells was approximately 40%, a fresh medium containing siRNA fragments or reagents was added. According to the different treatments, SY5Y cells were randomly divided into different groups, including the normal, NC, Reagent, SAMSN1‐F1, SAMSN1‐F2, and SAMSN1‐F3 groups. After 48 h, RT‐qPCR was performed to identify the interference efficiency, and the siRNA fragment with the highest interference efficiency was selected for subsequent experiments.2.11After culturing SY5Y cells for 5 days, the culture medium was removed in half, and a mixture solution  was added to each well of 6‐well plates. The cells were incubated for 15–30 min at normal temperature, adding 1 ml medium per well after 24 h. Images were collected via Leica AF6000 cell station (Leica) after 48 h, and cell numbers were calculated by choosing five randomly selected fields (200×). And the jamming effects were assessed by RT‐qPCR after 4 days of transfection.2.12Cells were seeded into 96‐well plates and cultured in an incubator. When the cell monolayer was covered with the bottom of the well, 20 μl of MTT solution was added to each well of the 96‐well plate, and the culture medium with MTT was incubated at 37°C for 4 h, and then the medium with MTT was aspirated. Each well was added with 100 μl dimethyl sulfoxide (Biosharp) solution, and then placed the 96‐well plate on an oscillator for 10 min. Finally, cell viability was calculated by and measured the absorbance value at 570 nm using a microplate reader.2.13Cells were seeded in 96‐well plates, images of nine fields were scanned for each time point, and then cells in these images were counted. Each experimental group had three multiple wells, and the cell count values of each group were averaged. With the cell number as the ordinate and time point as the abscissa, the cell growth curve of each group for 5 consecutive days could be drawn. The differences in cell growth curves among the experimental groups could directly represent the differences in cell proliferation and apoptosis.2.14t‐test was used for comparison between the two groups, one‐way analysis of variance was used for comparison between the three groups or more, least significant difference was used for comparing the difference between the two groups if the variance was homogeneous, Dunnett T3 was used for pairwise comparison if the variance was not homogeneous. p < 0.05 was regarded as statistically significant.Data were statistically analyzed utilizing SPSS 21.0 software (SPSS) and displayed as the mean ± SD. Independent‐sample 33.1p > 0.05). However, the scores were significantly elevated after HI compared with the sham group, especially at 12 h. A significant decrease in the score was also observed 24–48 h after HI . To assess the underlying role of HI‐induced injury, gene sequencing was performed to detect the DEGs in HI neonatal rats . Moreover, compared with the normal group, the SAMSN1 level in cortical fetal human cortical neurons was significantly enhanced in the OGD‐4 h group . The fold change of SAMSN1 in cells and neurons showed the same trends with the RT‐qPCR and sequencing results .We further evaluated the role of the F1–F3 interference sequences of SAMSN1 in vitro. The differentially relative expression among groups of normal, reagent, NC, F1, F2, and F3 reflected the extent to which the expression of SAMSN1 was downregulated. Meanwhile, the Cy3 labeled siRNAs were transfected into SY5Y cells and displayed red fluorescence expression in cells, manifesting the SAMSN1‐siRNA efficiently transfected into the SY5Y cells Figure . In addi3.5p < 0.05). Besides, the cell growth curve displayed that cell numbers exhibited an uprising trend 1–6 days after SAMSN1‐siRNA transfection, significantly increasing at 5 and 6 days in comparison to the Reagent group .MTT assay was used to detect cell viability after SAMSN1‐siRNA transfection. It was found that cell viability was obviously increased after the treatment of SAMSN1‐siRNA compared with the normal group  family, is thought to control inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity. It has been reported that inhibitory LILRBs play an essential role in innate immunity.5Overall, the expression of SAMSN1 was upregulated in neonatal rats following HI‐induced neurological injury and in cultured cells after OGD. Moreover, inhibition of SAMSN1 could significantly promote cell viability and cell survival with OGD. Our data revealed in NHIE, that suppression of SAMSN1 contributes to neuroprotection, which provides a potential theoretical basis for further research and treatment on NHIE.Yi‐Bo Wang contributed to the conceptualization, methodology, investigation, validation, writing original draft, review, and editing. Zong‐Jin Gan contributed to the statistical formal analysis and investigation, visualization. Jun‐Yan Zhang contributed to the statistical formal analysis and investigation, visualization. Somjit Wanchana contributed to software, supervision, and resources. Xi‐Liang Guo contributed to conceptualization, funding acquisition, methodology, project administration, and data curation.The authors declare no conflict of interest.The cortical brain tissues were harvested from a naturally aborted fetus provided by the First Affiliated Hospital of Kunming Medical University, which was approved by the Ethics Committee of Kunming Medical University on September 30, 2015 . The rats were fed following the guideline of the China Laboratory Animal Protection and Ethics Committee. The study was approved by the Ethics Committee of Kunming Medical University and the Animal Care & Welfare Committee of Kunming Medical University (no. KMMU2020001) and followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.


Following the publication of this article , concernchbP results appear more similar than would be expected from independent samples.The Fig 1A Bacterial lysate K96243 and Bacterial lysate chbP/pCHBP and 6h U937+bsaQ results appear to be more similar than would be expected from independent samples.The Fig 4A 6h U937+There appear to be repetitive patterns in the Fig 4A results.Burkholderia pseudomallei strain K96243 lysate and its isogenic chbP mutant in Fig 1A, as well as the lysates of the U937 cells in Fig 6A, are expected to look similar, as there is only a minor difference in predicted proteins. Underlying data were provided for the Fig 1A Bacterial lysate and Secreted protein panels. The underlying data provided for the Bacterial lysate results suggested that the panel was spliced during figure preparation, and the Bacterial Lysate K96243 lane did not appear to match the underlying data provided. The underlying data provided for the Secreted protein results showed that the results were not aligned to the marker lane appropriately; the series of bands aligned with the 95kDa marker in the published results instead appeared just below the 72kDa marker in the underlying data, and the series of bands aligned with just below the 17kDa marker in the published results instead appeared in line with the 10kDa marker in the underlying data.The corresponding author disagreed that the Figs 1A and 4A results listed above appear more similar than expected and commented that the protein profile of the The corresponding author was unable to explain the appearance of the repetitive features in the Fig 4A results, but they stated that there are also differences between the Fig 4A results listed above. The underlying data provided for the Fig 4A results did not explain the appearance of the repetitive features either, but the data provided appear to confirm the published results. However, upon examination of the underlying data provided for the Fig 4B results, additional concerns were raised that in the underlying data provided the backgrounds for the following lanes appear more similar than would be expected from independent data:BopE “U937+K9 3h” and CHBP “Uninfected U937”chbP 3h” and CHBP “U397+K9 6h”BopE “U937+chbP/pCHBP 3h” and CHBP “U397+chbP 3h”BopE “U937+bsaQ 3h” and CHBP “U937+chbP/pCHBP 3h”BopE “U937+BopE “U937+K9 6h” and CHBP “U937+K9 3h”chbP/pCHBP 6h” and CHBP “U397+chbP 6h”BopE “U937+PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels and underlying data that question the integrity of these data, the VM, MPS, and SK agreed with the retraction. PP and NJ did not agree with the retraction and stand by the article’s findings. SK apologizes for the issues with the published article. CVB, PK, KP, and JMS either did not respond directly or could not be reached.


In the end matter’s Supplement 1 table of contents and in Supplement 1, “eTable 1. Strengthening the Reporting of Observational Studies in Epidemiology Checklist” should have been deleted and all eTables renumbered accordingly. This article has been corrected.1In the Original Investigation titled “Restricted Mean Survival Time Analysis to Estimate SGLT2i–Associated Heterogeneous Treatment Effects on Primary and Secondary Prevention of Cardiorenal Outcomes in Patients With Type 2 Diabetes in Taiwan,”


The two moiré sites in the first layer WSe2 interfacing WS2 allow the formation of two different interlayer excitons, with the hole residing in either moiré site of the first layer WSe2 and the electron in the third layer WSe2. An electric field can drive the hybridization of either of the interlayer excitons with the intralayer excitons in the third WSe2 layer, realizing the continuous tuning of interlayer exciton hopping between two moiré sites and a superposition of the two interlayer excitons, distinctively different from the natural trilayer WSe2.Moiré superlattices of semiconducting transition metal dichalcogenides enable unprecedented spatial control of electron wavefunctions, leading to emerging quantum states. The breaking of translational symmetry further introduces a new degree of freedom: high symmetry moiré sites of energy minima behaving as spatially separated quantum dots. We demonstrate the superposition between two moiré sites by constructing a trilayer WSe 2 and monolayer WS2 host interlayer excitons localized at different moiré sites. Using an electric field, the authors tune the superposition between two moiré sites via hybridization with intralayer excitons in WSe2.The moiré lattice of trilayer WSe The inversion symmetry breaking in transition metal dichalcogenides (TMDCs) gives rise to a valley degree of freedom that is promising for valleytronics and quantum information science based on valley-spin2, which can be accessed through chiral light.Design and control of symmetries can lead to symmetry-protected states that are promising to revolutionize the field of quantum materials. For example, breaking the inversion symmetry or time-reversal symmetry can lead to Weyl semimetals9, which are constructed through twisted TMDCs with a lattice mismatch or twist angle, enables spatial control of the excitons in two-dimension (2D) with the tunable periodicity of 1–10 nm and ushers in unprecedented opportunities in engineering electrons and excitons, leading to intriguing correlated electronic states15, the array of quantum emitters, and correlated exciton states resulting from flat excitonic band18.The recent emergence of semiconducting TMDC moiré superlattices20 that can confine electrons and excitons, as schematically shown in Fig. 19. As a result, coupling and hybridization of these high symmetry points will usher in new venues toward quantum information storage and processing. However, unlike the energy degeneracy of different valleys (K and K’), the energy barrier between different moiré sites (on the order of 10 s’ meV), along with their spatial separation, greatly suppresses the direct coupling between these moiré sites.Translation symmetry breaking in TMDC moiré superlattices introduces a new degree of freedom: moiré sites, the high symmetry points in a moiré supercell, which can be local energy minima and act as quantum dots21. In an angle-aligned trilayer WSe2/monolayer WS2 heterojunction (3L WSe2/1L WS2), new types of interlayer excitons emerge, with holes residing in the first WSe2 layer either trapped in moiré A or B site  25. XA is redshifted linearly for both n and p doping in a symmetric fashion, with a slope of ~1.3 meV/1012 cm−2. 2 layer, which have two degenerate modes as schematically shown in Fig. 2, and from WSe2 to WS2 in 3L WSe2/ 1 L WS2.). A is a less pronounced resonance XA’ with a larger slope (2.7 meV/1012 cm−2). XA’ is likely the exciton resonance of the middle (second) layer WSe2 and is not the focus of this work , which is only observable in angle-aligned 3L WSe2/1L WS2 heterojunctions but absent in heterojunctions with large twist angles  is likely due to the built-in electric field on WSe2 layers arising from the electron accumulation in WS2. We leave the related discussion in Supplementary Information Section 2 of the 3L WSe2/1L WS2 moiré heterojunction, with their schematics shown in Fig. n = 1). The nature of both resonances are revealed in our later discussion of the electric field dependence study.In the optical reflectance contrast spectra of 3L WSeion Fig. , there iSe2 Fig. , with a 2 device is shown in Fig. 2/WS2 (0.7 nm)7, confirming that the electron and hole of interlayer exciton occupy the two outside WSe2 layers in a natural trilayer WSe2.The electric field-dependent reflectance contrast spectra of the trilayer WSe2, there is significant tunneling of holes between the first and third layer WSe2 as they have the same valley-layer pseudo spin, allowing the hybridization of the interlayer excitons with the intralayer excitons in either the first or third WSe2 layer27, as schematically shown in Fig. The level avoiding at the intralayer exciton A (~1.70 eV) in Fig. A (black dotted line) can be used to well fit the observed hybridized spectra (red and blue dashed lines). From the fitting, we extract the coupling strength to be 10.7 ± 0.3 meV, larger than the linewidth of the hybridized exciton (~ 9.0 ± 0.3 meV). The scenario of the negative electric field is similar, where the other interlayer exciton mode, A) when the energy of the two excitons is tuned to resonance via the electric field. It is worth noting that we ignore the conduction band hybridization of the first and third layer WSe2, which is theoretically predicted to be nonzero but orders of magnitude smaller than the holes27. The neglection of the conduction band hybridization is also justified by the electric-field-dependent reflectance contrast spectra of 3 L WSe2/ 1 L WS2, which is asymmetric about positive and negative electric fields  as determined by the second harmonic generation (SHG) spectra  of 3 L WSe2/ 1 L WS2 in the dual-gate configuration, with an intentionally misaligned angle (20-degree) between WSe2 and WS2 layers. The electric-field-dependent reflectance contrast spectra  flakes are exfoliated on silicon chips with 285 nm thermal oxide. The thickness of BN flakes was determined by atomic force microscopy (AFM). The layer numbers of WSe2 flakes were identified by optical contrast with the assistance of second-harmonic generation (SHG). Top BN and bottom flakes with equal thickness were intentionally used for devices D1, D2, and D3. The polycarbonate (PC)/ polydimethylsiloxane (PDMS) stamp was used to pick up TMDC monolayer and other flakes sequentially. The alignment of each layer is achieved under a home-built microscope transfer stage with the rotation controlled with an accuracy of 0.02 degrees. The PC is then removed in the chloroform/isopropanol sequence and dried with nitrogen gas. The final constructed devices were annealed in a vacuum (<10−6 torr) at 250 °C for 8 hours.We used the same dry pick-up method0 was obtained by fitting the reflectance spectrum at high hole-doping level with a polynomial function for each measured spot  and collect the optical signal into a spectrometer (Princeton Instruments). The reflectance contrast measurement was performed using a supercontinuum laser source (YSL photonics). A relative flat reflectance background R32.The density of carriers introduced by the electrostatic gating is given by Supplementary InformationPeer Review FileSource Data


Dr Belay’s name should be listed as Brook Belay, MD, MPH. The first sentence of the Findings section of the Key Points should be the following: “In this cohort study of 13 381 050 US patients with a BMI of 25 or higher, the annual probability of 5% or greater weight loss was low (1 in 10) but increased with higher initial BMI.” This article has been corrected.In the Original Investigation titled “Probability of 5% or Greater Weight Loss or BMI Reduction to Healthy Weight Among Adults With Overweight or Obesity”


The pearl‐like KB branch chains encircle the NVFP (p‐NVFP), the segregated particles possess promoted overall conductivity, balanced charge, and modulated crystal structure during electrochemical progress. The p‐NVFP obtains significantly enhanced ion diffusion ability and low volume change (2.99%). Meanwhile, it delivers a durable cycling performance (87.7% capacity retention over 5000 cycles at 5 C) in half cells. Surprisingly, the full cells of p‐NVFP reveal a remarkable capability of 84.9 mAh g−1 at 20 C with good cycling performance (capacity decay rate is 0.016% per cycle at 2 C). The structure modulation of the p‐NVFP provides a rational design on the superiority of others to be put into practice.Based on the favorable ionic conductivity and structural stability, sodium superionic conductor (NASICON) materials especially utilizing multivalent redox reaction of vanadium are one of the most promising cathodes in sodium‐ion batteries (SIBs). To further boost their application in large‐scale energy storage production, a rational strategy is to tailor vanadium with earth‐abundant and cheap elements , reducing the cost and toxicity of vanadium‐based NASICON materials. Here, the Na 3.05V1.03Fe0.97(PO4)3 (NVFP) like wearing “pearl,” making the segregated particles link to access the overall conductivity and activating the unusual structural distortion of FeO6 in NVFP during electrochemical progress, thus stimulating plenty of Na+ de/intercalation at plateau region with reversible phase transition and acquiring a favorable rate performance and durable cycling lifespan under fine volume variation.Ketjen Black (KB) branch chains encircle the Na Nevertheless, developing more promising supplements has been considered an urgent need for boosting grid‐scale electric storage applications in recent decades, as the development of LIBs is severely limited by the limited stock of the raw material and the growing cost. Owing to the widespread abundance and low cost of sodium resources, as well as similar working principles as LIBs, sodium‐ion batteries (SIBs) display great potential in energy storage systems, especially in energy‐intensive environments that focus on economic benefits. Nevertheless, the primary challenge of SIBs is to provide efficient electrochemical performance comparable to LIBs, particularly in energy density and cycling stability.With the robust development of energy storage systems, electric vehicles and electronic devices based on powerful smart grids are also continuously improving, and gradually shifting to the mass market, in turn, bringing more demands for energy storage. Among diversified energy storage systems, devices like rechargeable batteries have become one of the most promising choices for energy reserves with favorable energy conversion efficiency and convenient maintenance.1 At p owing to their intrinsic structural advantages, polyanionic compounds are always regarded as one of the most fascinating cathodes in SIBs: the inductive effect of polyanionic groups makes high operating voltage possible, thereby obtaining relatively high energy density; the robust frameworks are able to diffuse Na+ rapidly and provide long‐term structural stability, which is favorable for high power density, operation reliability and security. Among the polyanionic compounds, sodium superionic conductor (NASICON) possesses a 3D robust framework, buffering volumetric expansion during phase transition process and strong covalent bonding with predominant ionic conductivity for the migration of Na+. Eventually, NASICON‐type cathodes like Na3V2(PO4)3 (NVP) attract colossal interest, benefitting from their high safety, and high thermal and structural stability. NVP has been extensively investigated because of its promising two‐electron reaction, high sodium ion mobility, high working voltage plateau of about 3.4 V, bringing it into the limelight of large‐scale energy storage. However, the toxicity and cost of vanadium have become the bottleneck which limit its practical application.Electrode materials as sodium hosts play a vital role in SIBs, not only as the source of energy but also as the cornerstone of maintaining stability. Commonly, cathode materials of SIBs include layered, polyanionic compounds, Prussian blue and organics4 owin The regulation of Mn3+/Mn2+, Fe3+/Fe2+ and Ti3+/Ti2+ redox centers has brought a series of cathode materials, which show promising features in terms of enhanced output voltage and specific capacity. The iron‐based compounds in V‐substituted cathodes have better cycling stability compared with Mn‐based counterparts with the Jahn‐Teller effect and possess a higher redox potential of Fe2+/Fe3+ (2.4 V) than that of Ti‐based substitution materials (Ti3+/Ti4+ (2.1 V)). Therefore, in the wide selection of transition metals, the choice of Fe substitution of the V site is considered to be the best choice for reducing toxicity and cost compared to NVP as well as maintaining fine electrochemical properties.Thanks to the flexible tunability of the NASICON structure, different choices of transition metal sites have been verified by the replacement of V with various elements in NVP.9 The + and reducing the content of vanadium, Na4FeIIVIII(PO4)3 emerges in the class of mixed‐metal phosphates, which is expected to display great performance through the successive activations of the Fe3+/Fe2+, V4+/V3+ and V5+/V4+ redox couples. Although the systematic study and attempts have been implemented in preparing Na4VFe(PO4)3 directly, the achievements often fall short of expectations, tending to form the sodium‐deficient composition with mixed valence Fe2+/Fe3+ 3 and Na3.36FeV(PO4)3 via a traditional sol‐gel route). The absence of Na+ in the sodium‐deficient composition needs to be compensated by the free Na+ in the electrolyte to obtain the Na4VFe(PO4)3 with electrochemical cycles. What's more, a systematic study of Na3+xFexIIV2‐xIII(PO4)3 is also to produce secondary phases (mainly NaFePO4) when x > 0.5 in solid‐state synthesis. It is difficult to prepare a pure‐phase Na4VFe(PO4)3 target as many secondary phases appeared in the samples through direct synthesis. Recently, an effective way was implemented by ball‐milling assisted sol‐gel method combined with heat treatment in preparing the pure‐phase Na4VFe(PO4)3. In such a synthesis process, the amorphous carbon single layer coated on the surface of Na4VFe(PO4)3 was only with 3 nm thickness and assist Na4VFe(PO4)3 to obtain a typical rhombohedral NASICON‐type structure without other impurities. The thin carbon layer from the carbonization of glucose not only improves the electronic conductivity of Na4VFe(PO4)3 materials intrinsically but also influences crystal structure to ensure the stable state of Fe in Na4VFe(PO4)3. In addition, a handy way is using Na3FeIIIVIII(PO4)3 as a precursor by electrochemical synthesis to obtain pure Na4FeIIVIII(PO4)3. And the Na3FeV(PO4)3 itself is easy to synthesize and has a decent electrochemical performance with two voltage plateaus at 3.3 and 2.5 V. Despite the local environments and valence states of Fe and V being different in Na3FeV(PO4)3 and Na4VFe(PO4)3, they similarly deliver an initial discharge capacity of about 100 mAh g−1 based on a two‐electron reaction, and Na3FeV(PO4)3 shows a durable capacity retention even at 10 C with subtle volume change. Therefore, Na3FeV(PO4)3 has advantages in terms of manufacturing technique, specific capacity and cycle durability compared with Na4VFe(PO4)3 based on the V4+/V3+ and Fe3+/Fe2+ redox reaction.Considering more accumulation of Na couples.12 Alt3.05V1.03Fe0.97(PO4)3 (NVFP) by partially replacing V with Fe. Moreover, besides the carbon coating produced from the sol‐gel process, the spherical highly conductive Ketjen Black (KB) with a unique shape of branched chains is also selected to improve the electronic conductivity, balance the valence state and modulate the main structure in the initial electrochemical process. The branched chains like “pearl” encircling independent NVFP particles (p‐NVFP) provide more conductive paths, greatly improving the current density and battery capacity, and further extending the service time of the battery. The particular structure of p‐NVFP presents the performance with favorable capacity (106.8 mAh g−1 at 0.2 C), remarkable high‐rate capacity (87.8 mAh g−1 even at 15 C) and long cycling (stable operation exceeds 5000 cycles at 5 C). Furthermore, we elucidate the effects of active branch chain infusing on the crystal structure distortion by using in situ X‐ray diffraction (XRD) and ex situ X‐ray photoelectron spectroscopy (XPS). The ionic migration kinetics in such a special structure is to be investigated by the steady operation of p‐NVFP by cyclic voltammetry (CV) curves and galvanostatic intermittent titration technique (GITT). Stimulating from the rapid kinetics at voltage plateaux, the full cells of p‐NVFP reveal a remarkable capability with high capacity retention of 84.9 mAh g−1 at 20 C and durable cycling performance (capacity decay rate is with 0.016% per cycle at 2 C). Such a simple but effective structure design buffers the impact of current on the host materials by the decentralized transmission network of “pearl,” and mixed state of p‐NVFP makes the solid‐solution reaction adequate in sodium storage mechanism, which contributes a great insight for other systems with cost‐effective elements for large‐scale energy storage applications.Herein, in this work, we engineer an extraordinary NASICON material Na22.1FigureFigureThe p‐NVFP particles were synthesized via ball‐milling assisted sol‐gel method in the Experimental Section. The same production process was used in preparing NVFP (without joint of KB) and p‐NVFP (design with branch chains like “pearl” of KB). The morphology and microstructure of NVFP‐based materials were monitored by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in Figures R_3c with NVP. While there is a trace of undesirable NaFePO4 in NVFP like in the previous research, it disappears after ball‐milling with KB followed by annealing at high temperature. And the main phase NVFP is consistent with the standard card of NVP (PDF#00‐062‐0345). Simultaneously, the crystal structure of p‐NVFP is derived from the Rietveld refinement of the XRD as illustrated in Figure  The octahedra are randomly filled by Fe and V ions. The Fe/VO6 octahedra connected with PO4 tetrahedra by corner‐sharing, establishing a 3D anion structure for accommodating Na+ flux. With different oxygen environments, Na+ at two differentiated sites possess different coordination in the interstitial space. Analyzed by Raman spectroscopy, two characteristic carbon peaks for NVFP‐based materials, which were located at about 1380 cm−1 and 1590 cm−1, corresponding to the disordered carbon and graphitized carbon (D band and G band), respectively. And the graphitized proportion is measured by the relative intensity of two (ID/IG), which originated from carbon components (citric acid and KB). The ID/IG values of NVFP, p‐NVFP are 1.33 and 1.01, respectively. Moreover, the carbon contents of NVFP and p‐NVFP materials are 1.28 wt% and 5.76 wt%, measured with the elemental analysis are shown in Figure −11 S cm−1 compared to 6.37 × 10−12 S cm−1 of NVFP according to Table  The spectrum of V 2p splits into two sharp peaks located at about 523.42 eV and 516.40 eV, corresponding to the electrons in V 2p1/2 and V 2p3/2, respectively, confirming trivalent V and in good agreement as expected. The high‐resolution Fe 2p XPS spectra of p‐NVFP are split into five peaks with one satellite peak, two pairs of sharp peaks centered at about 711.96 eV and 726.45 eV, 709.6 eV and 725.69 eV, corresponding to the electrons in Fe3+ and Fe2+, respectively, confirming trivalent and partly divalent in the p‐NVFP. The molar ratio of compounds in p‐NVFP is 3.05: 0.97: 1.03 for Na, Fe and V by inductively coupled plasma spectroscopy (ICP‐OES) chemical analysis, which is well matched for the mixed state of Fe. Finally, based on all the above results, it can be inferred that the p‐NVFP material is a mixed state Fe‐substituted phosphate compound for NVP.As shown in Figure with NVP.13 Whi2.2Figure3+/V4+ redox reaction at high voltage plateau and takes off more Na+ in p‐NVFP than those in NVFP, thanks to the collection toward current of KB branch chain. As a result, the capacity of p‐NVFP cells reaches 106.8 mAh g−1 in first discharge progress and the initial Coulombic efficiency (CE) of the two is above 100%, which corresponds to consuming some Na+ in electrolyte. However, the CE of the two remains around 100%, without continuously consuming additional Na+ during subsequent cycles at the same current. And the dQ/dV comparison also demonstrates that the reduction peaks of p‐NVFP are higher than the counterpart around 3.45 V of NVFP. Similarly, the anodic peak (A2) at high plateau in the first cycle is sharper than other anodic peaks and located at higher potential (3.50 V), originating from the adequate V oxidation during Na+ de/intercalation in CV curves of Figure + migration around the Fe/VO6 octahedra under the influence of “pearl” structure. Except for the first cycle, the respective CV curves coincide well in the subsequent cycles, implying the stable effect of the two on the reversible structure. Such a differentiation would make an effect on the subsequent electrochemical processes, which will be discussed in the following section.To explore the Na storage ability of NVFP‐based materials, the electrochemical tests were implemented with Na plate as a reference electrode in the voltage window of 1.9–3.8 V. And the first two galvanostatic charge/discharge (GCD) curves of NVFP‐based materials were compared in −1). The revert capacity up to 105.7 mAh g−1 was obtained when the current density returned to 0.1 C. After several cycles at 0.1 C, the rate performance at 0.2 C elevated slightly and reached 108.2 mAh g−1. Admittedly, the voltage differentiation induced by “pearl” structure makes the polarization obviously decrease, especially at high current density in Figure + adequately even in fast charge process, but also maintains stable intercalation reaction, thus the electrochemical property of p‐NVFP is superior to NVFP even with the same redox reaction. Benefitting from the improved reversibility, the cycling stability of p‐NVFP also gets promoted as shown in Figure In addition, the potential differentiation of p‐NVFP also exists in the initial cycling according to Figure 2.3Figure All the XRD peaks almost return to their original Bragg positions and intensities after one charging/discharging cycle, indicating excellent reversibility of insertion/extraction with a single‐phase solid solution reaction. Figure +) for the first two cycles. The previous research showed that there was a continuous decrease in the lattice parameter a, while the c parameter increased during the charge process. However, the lattice parameter c goes down in the first charge process and rises first and then decreases in the second charge process, which seems to produce unusual structural distortion compared to NVP. The structure evolution goes further with the shrink of subsequent transport channels along c axis, thus making more Na+ extract from the main structure, which corresponds to more capacity at high voltage plateau in charge process. It is speculated that the peculiar decrease of c parameter could be likely to relate to the contraction of FeO6 octahedrons with the oxidation of Fe2+ to Fe3+. The changes in valence‐state and relative contents of Fe and V provide direct evidence to comprehend the redox process of the p‐NVFP during charging/discharging process. As shown in Figure Utilizing the in situ XRD and ex situ XPS technologies, the sodium storage mechanism of p‐NVFP in the initial two cycles was elaborated in 3+/V4+ at 3.45 V, the trace of Fe2+ in p‐NVFP would generate surface oxidation to Fe3+ . At the same time, the XPS spectra of C1s at point b (3.46 V) demonstrate a shift toward low binding energy, differing from oxidation in charge process and implying the interaction with FeO6. The valence‐state changes between point a  and point b (3.46 V) in Figure 2+ in p‐NVFP along with the structural adjustment. Through this modulation, p‐NVFP obtains the preferable buffer ability of Na+, whose volume change is significantly lower than other NASICON materials in Figure  In summary, the “pearl” structure makes the Fe2+ of NVFP take surface oxidation, assisting more deintercalation of Na+ along c axis in the initial structural adjustment and stimulating the subsequent V3+/V4+ redox completely.Before the potential of Vn Figure 21 In 2.4Figure−1. Figure Ip) as a function of scan rates square root (𝜐1/2), suggesting that the Na+ extraction/insertion mechanism is controlled by the diffusion process. Calculated by CV profiles, the Dapp,Na values of p‐NVFP are about tens to hundreds of times higher than those of NVFP at respective redox reactions in Figure + de/intercalation with improved performance by “pearl” structure. Figure τ) and the corresponding cell voltage (E) for electrode material during the second cycle. And the fitting results between E and the square root of galvanostatic time (τ1/2) during a single GITT titration process are shown in Figure Dapp,Na values of p‐NVFP in the voltage plateau region are also higher than those of the NVFP electrode, which is consistent with the results of the CV tests. In particular, the diffusion at the low plateau region of Fe2+/Fe3+ in NVFP is far below the average Dapp,Na value (dotted lines in each picture) and significantly behind p‐NVFP . In addition, the Dapp,Na value drops down markedly between the plateau regions in NVFP, which is obviously different from p‐NVFP, whether is in charging/discharging process. The reduction of the difference is related to the unusual structural distortion in p‐NVFP as mentioned in Figure Dapp,Na value of low potential plateaux. Hence, the excellent kinetic properties further explain the reasons for the improved electrochemical performance and cycle performance, which are caused by the modulation of branch chains in the “pearl” structure.Furthermore, the kinetic process of p‐NVFP with voltage differentiation was performed to study the electrode reaction by CV tests at different sweep rates and GITT. As shown in Figure−1 at 0.1 C and retains 84.9 mAh g−1 even at 20 C in Figure  Impressively, the cycling stability of the full‐cell is as excellent as that of the p‐NVFP half cell in Figure −1, and delivers capacity retention of 92% after 500 cycles at 2 C and the capacity decay rate reaches to 1.5‱ per cycle. Figure Based on the above results, the full cells using hard carbon (HC) as anode were assembled and the capacity was controlled by the cathode (N/P = 1.1), schematically illustrated in 3−1 at 0.2 C), remarkable high‐rate capacity (87.8 mAh g−1 even at 15 C) and long cycling (stable operation exceeds 5000 cycles at 5 C). Stimulating from the rapid kinetics at voltage plateaux, the full cells were assembled with the commercial HC. p‐NVFP reveals a remarkable capability with a high capacity of 84.9 mAh g−1 at 20 C and durable cycling performance (capacity decay rate is 0.016% per cycle at 2 C). Such a simple but effective structure design buffers the impact of current on the host materials and makes the solid‐solution reaction adequate in sodium storage mechanism, which contributes a great insight for other cost‐effective systems for large‐scale and durable cycling lifespan in energy storage applications.The effects of active branch chain infusing on the crystal structure distortion were analyzed by in situ XRD and ex situ XPS, and ionic migration kinetics were examined by CV curves and GITT to have an insight into the structure modulation of p‐NVFP. The branch chains like “pearl” encircling independent p‐NVFP provide more conductive paths, greatly improving the current density and battery capacity, and further extending the service time of the battery. The particular structure of p‐NVFP presents the performance with favorable capacity (106.8 mAh g43)3∙9H2O, NH4VO3, and CH3COONa were added with a certain amount of distilled water into the round‐bottomed flask. Then, a saturated citric acid solution was added dropwise to the round‐bottomed flask as carbon source and complex agent and stirred for 30–50 min to make the solution evenly mixed. And then added NH4H2PO4 into the above solution while stirring. Subsequently, the distilled water was evaporated at 80 °C to form a wet gel. The resulting wet gel was dried at 100 °C overnight in a vacuum oven to form a dry gel. After grinding the dry gel, it was pre‐calcined at 350 °C for 4 h in the reducing atmosphere. The samples after pre‐calcination were ball‐milled and calcined at 500 °C for 8 h to obtain the final product NVFP. The synthetic raw material is denoted as NVFP, and the ball‐mill product with adding 4wt% KB is denoted as p‐NVFP.The NVFP cathode materials were synthesized by a sol‐gel method. First of all, a stoichiometric amount of Fe(NOα radiation (λ = 0.15418 nm) in the scan range (2θ) of 10–80°. The morphologies information, element distribution and particle size of each compound were obtained by using the scanning electron microscope  and high‐resolution transmission electron microscopy  equipped with an energy dispersive X‐ray spectrometer (EDX). The Raman spectra were obtained by using a laser confocal micro‐Raman spectrometer (JYHR‐800 Lab Ram) with an excitation laser beam wavelength of 488 nm. The X‐ray photoelectron spectroscopy (XPS) analysis was obtained by ESCALab 220i‐XL electron spectrometer from VG Scientific with 300 W Al Kα radiation to determine the valence states of the prepared materials. And the carbon content was measured by CHN Elemental analyzer (Eurovector‐EA3000). Inductively coupled plasma spectroscopy (ICP) analysis was obtained by Prodigy. Electronic conductivity testing was measured by four‐terminal powder resistivity tester (ST2722‐SZ).The X‐ray diffraction (XRD) dates were collected in an X‐ray diffractometer  with Cu K−2. The CR2032 type coin cells consisted of the prepared electrode as the cathode, the metal sodium plate as the counter and reference electrode, the glass microfiber membrane (Whatman) as the separator, and electrolytes (1 mol L−1 NaClO4 dissolved in the ethylene carbonate (EC) and propylene carbonate (PC) solution with a volume ratio of 1:1 and 5 vt% fluoroethylene carbonate (FEC) as an additive) were assembled. The galvanostatic charge/discharge (GCD) tests at different current densities were implemented on the battery testing systems (NEWARE CT‐4000). Cyclic voltammetry (CV) tests were obtained on a Versa STAT 3 electrochemical workstation in the potential range of 1.9–3.8 V. The current density of the galvanostatic intermittent titration technique (GITT) tests was 0.05 C in the potential range of 1.9–3.8 V, the duration time of each current pulse was 1 h, and the resting time was 6 h. The p‐NVFP cathode was matched with the hard carbon (HC) anode in a glove box to assemble full cells, and their electrochemical performance was tested within the same potential window as half cells. The specific capacity of the full cell was calculated based on the mass of the active material in the cathode. Before assembling the full cells, presodiation for the HC anode was performed to activate the material and stabilize the electrode surface. Specifically, by chemical presodiation, electrolyte was added to the anode material and contacted with the metal sodium directly for 30 min.The cathode electrode consisted of the active material NVFP, the conductive agent KB and the binder carboxymethylcellulose (CMC) with a mass ratio of 8:1:1. The slurry was uniformly coated on the Al foil. The electrodes were dried in a vacuum oven at 60 °C for 10 h. The loading mass of active material on the final working electrode is about 0.8–1.3 mg cmip) and the square root of the sweep rate (v1/2) derived from the CV curves. Of all the NVFP‐based composites can observe a good linear fitting result clearly, proving that de/intercalation of Na+ during the charge‐discharge process is controlled by diffusion. Therefore, the corresponding  in the electrode material can be calculated by the following Randles‐Sevcik formula:ip is the peak current density, n is the number of electrons transferred per reactant molecule during the charge‐discharge process of the NVFP electrodes (n = 2 for the NVFP phase), A is the electrode surface area of 1.13 cm2, and C0 is the concentration of Na ion in the crystal, and v is the sweep rate.The fitted relationship between the peak current density  were further calculated by GITT tests to evaluate the sodium ion diffusion kinetics of the three NVFP‐based composites. The Dapp,Na can be calculated by the following formula:mB is the mass of the NVFP material, VM is the molar volume, MB is the molar molecular mass, τ is the time for an applied galvanostatic current (3600 s) during each titration, S is the electrode surface area (1.13 cm2), and L is the average radius of the material particles, ΔEs and ΔEτ are the quasi‐equilibrium potential and the change in cell voltage during the current pulse, respectively.The apparent sodium ion diffusion coefficients Dapp,Na weThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Streptomyces violens was identified and represents the second known sesterterpene synthase from bacteria. Isotopic labelling experiments in conjunction with DFT calculations were performed that provided detailed insight into its complex cyclisation mechanism. Enzyme engineering through site‐directed mutagenesis gave access to a high‐yielding enzyme variant that provided six additional minor products and the main product in sufficient quantities to study its chemistry.The sesterviolene synthase from  Streptomyces violarus is the second bacterial sesterterpene synthase to be described. The enzyme generates one main and several side products whose mechanisms of formation were studied in isotopic labelling experiments and through DFT calculations. Enzyme engineering allowed isolation and structural characterisation of the side products and gave access to the main product in sufficient quantity to study its chemistry.Sesterviolene synthase from  These reactions are multistep cascades via cationic intermediates that are generated in the case of class I terpene synthases (TS) by the abstraction of diphosphate,S. violens showing 43 % sequence identity to SmTS1 was selected for further study through gene cloning and expression in Escherichia coli. The purified enzyme  converted GFPP into one major sesterterpene hydrocarbon beside a few trace compounds , while GPP, FPP and GGPP were not accepted. The main product was isolated (3 % yield from GFPP) and structurally characterised by NMR spectroscopy , resulting in the structure of sesterviolene A (1), which represents a novel skeleton (Scheme 1 was thus identified as Streptomyces violensSesterviolene Synthase (SvSS).A phylogenetic tree constructed from 3278 amino acid sequences of bacterial TS homologues Figure S1) identified a few candidate enzymes that are closely related to SmTS1. The TS homologue from  identifi1 proceeds through a 1,15‐ and 14,18‐cyclisation with a 1,5‐hydride shift from C12 to C19 towards B (the reasoning for cation A is discussed below). A subsequent 6,10‐cyclisation to C, 1,2‐hydride shift to D and 6,11‐cyclisation lead to E, which further reacts through 2,12‐cyclisation to F and 1,3‐hydride shift to G. Such 1,3‐hydride shifts are possible in terpene biosynthesis, but only for trans‐fused ring systems.H and opening of the cyclobutane ring result in I, which can undergo ring contraction to J and deprotonation to 1.The proposed cyclisation mechanism from GFPP to 1 was experimentally investigated through isotopic labelling experiments (Table S3). For this purpose, all 25 isotopomers of (13C1)GFPP, generated enzymatically with GFPPS from S. mobaraensis1 with incorporation of 13C‐labelling at the expected positions in all cases . Specifically, the obtained data were in agreement with the skeletal rearrangement from I to J, disconnecting C11 and C12, and revealed high face selectivity for the 1,5‐hydride shift from C12 to C19 in the conversion of A into B, as indicated by the fact that the labelling from C20 and C21 was not distributed over the two geminal Me groups in the product. The 1,5‐hydride shift from A to B was investigated using (7‐13C)GPPE)‐ or (Z)‐IPP13C NMR analysis showed a triplet for C19 (13C−2H spin coupling) in the experiment with (E)‐IPP, revealing specific migration of the hydrogen atom derived from 4HE of IPP . The 1,2‐hydride migration from C to D was explored using GGPP1 in the 13C NMR spectrum . Due to peak broadening of the signal for this carbon atom at 298 K, the triplet was only observable at higher temperature (343 K). The 1,3‐hydride shift from C12 to C3 in the step from F to G was investigated through conversion of GPP with IPP and (3‐13C)IPP with GFPPS and SvSS. This combination of substrates yields mixtures of isotopomers, but only the four isotopomers in which the fifth isoprene unit of GFPP is 13C‐labelled from (3‐13C)IPP are relevant for the detection of C3 by 13C NMR spectroscopy (Scheme S3), while four more isotopomers in which the fifth isoprene unit is deuterated from IPP do not result in a strong signal for C3 (not shown). Only if the third unit of GFPP is derived from IPP, C12 is deuterated and the 1,3‐hydride shift becomes visible by a triplet signal for C3 , but not if the third unit arises from (3‐13C)IPP . Finally, the terminal deprotonation step to 1 was investigated with (R)‐ and (S)‐(1‐2H)‐GPPS)‐(1‐2H)‐GPP . Because of the inversion of configuration at C1 of oligoprenyl diphosphates in the elongation with IPP,pro‐S hydrogen atom in J (top face in Scheme The cyclisation mechanism to 1 was determined through a stereoselective deuteration approach using DMAPP and (R)‐ or (S)‐IPP , and (E)‐ or (Z)‐IPP . With these substrates, stereochemical anchors of known configuration are introduced into 1 that allow determination of its absolute configuration by resolving the relative configuration of the naturally present stereogenic centers in 1 with respect to these anchors. The additional 13C label serves as a highly sensitive probe for analysis through HSQC spectroscopy. The results revealed the absolute configuration of 1 as shown in Scheme The absolute configuration of Site‐directed mutagenesis of terpene synthasesTo gain further insight into the active‐site architecture of SvSS, an enzyme model was created using AlphaFold2 . Under improved incubation conditions with the addition of β‐cyclodextrin, 150 mg of GFPP were converted with the D305N variant to yield 20 mg of 1 (22 % yield). Moreover, the production of the minor compounds already observed with wildtype SvSS was also increased , allowing their isolation and structure elucidation . The newly obtained compounds 2–7 were named sesterviolenes B–G . The formation of 5 and 6 must proceed with transannular proton or hydride shifts, similar to the reactions observed previously with the sestermobaraene synthase SmTS1.7 is the C18 epimer of 6, pointing to a slightly changed conformational fold of GFPP in the initial cyclisations.Biosynthetically, 5–7 in conjunction with a deuterium kinetic isotope effect that further lowered their yields, trials to follow the transannular hydrogen migrations in their biosynthesis by isotopic labelling failed. Therefore, DFT calculations were performed to further investigate the cyclisation mechanism of SvSS . Along the main pathway to 1  barriers or even barrierless, with the highest TS barrier found for the rearrangement from G to H. Notably, the cyclopropanation from D to E was found to be barrierless, and the ring contraction from I to J was associated with a very low TS barrier . Low TS barriers were also computed for the transannular hydrogen shifts in the biosynthesis of 5–7, showing the feasibility of the proposed steps (Scheme S4). The calculations could not resolve the question whether 4 is formed from J through two sequential 1,2‐hydride shifts or a single 1,3‐hydride migration, as the TS barriers for these pathways were, at 10.78 kcal mol−1 and 10.96 kcal mol−1, very similar. An isotopic labelling experiment using (2‐2H)GPP and (4‐13C)IPP with GFPPS and SvSS allowed us to distinguish between these alternatives, indicating a sequence of two 1,2‐hydride shifts by a moderate deshielding (Δδ=−0.08 ppm) for C12 caused by a deuterium at the neighbouring carbon atom C13 . Also, the loss of deuterium in the formation of 3 was in line with this model .Because of the low production of 1 obtained with the D305N variant allowed us to study the chemistry of this sesterterpene , which was slowly further converted into sesterviolene epoxide A (9) under these conditions . Potentially, 4 could form the same autoxidation products, but for this compound no changes were observed after prolonged standing in air. The different behaviour of these sesterterpenes may be explained by a release of strain from 1 resulting from a steric interaction of the iPr group with Me‐23 that is less pronounced in 8 (Scheme The high yields of e Scheme A. Intere1 with meta‐chloroperbenzoic acid (mCPBA) also proceeded with formation of a conjugated diene system and likely subsequent epoxidation. From the complex product mixture, the two bis‐epoxides sesterviolene epoxide B (10) and C (11) were isolated , in which not only were epoxide functionalities introduced, but the carbon skeleton was oxidised. In 10, the 10,11‐ and the 14,18‐epoxide functionalities were installed from the sterically less hindered sides, with the 14,18‐epoxide showing opposite configurations to those in 9. The small oxidant O2 may attack 1 from the less accessible side, leading to the thermodynamic product 9. The mCPBA‐mediated formation of 10 and 11 from 1 may proceed via intermediate 8. This hypothesis was tested by the oxidation of 8 with mCPBA, which resulted in the same mixture of 10 and 11 as obtained from 1 . Furthermore, the epoxidation of 9 yielded sesterviolene epoxide D , a stereoisomer of 10. Interestingly, the reaction of 4 with mCPBA gave the expected bis‐epoxide sesterviolene epoxide E , showing that in 4 with a saturated C12/C13 bond, the lower side of the C14/C18 double bond is more easily accessible. Besides these oxidations, 1 was treated with N‐bromosuccinimide (NBS), which resulted in a low yield of 10‐bromosesterviolene A .The treatment of Streptomyces violarus. The enzyme produces one main compound besides several other sesterterpene hydrocarbons, but their production was too low for compound isolation. Therefore, enzyme engineering through site‐directed mutagenesis based on an AlphaFold2 enzyme model was used to gain access to an enzyme variant (D305N) that showed much higher expression levels and better catalytic performance. The deeper reason for this intriguing effect and a possible catalytic function of the D305 residue are currently unclear and will require future structural work on SvSS. Not ony did the D305N enzyme variant enable isolation of the minor side products for structure elucidation, showing the high potential of structure‐model‐based enzyme engineering, but the main compound was made accessible in good yields, thus enabling its chemistry to be studied. Sesterviolene A is notoriously unstable and readily oxidised in air, with installation of a conjugated double bond that presumably releases strain from 1, followed by spontaneous epoxidation. Treatment with mCPBA caused similar reactions, but epoxidation at C14/C18 proceeded from the opposite site, and resulted in further oxidations. Similar to the sestermobaraene synthase from S. mobaraensis, SvSS is genetically clustered with a prenyltransferase likely for GFPP biosynthesis that shows 49 % identity to the characterised GFPPS from S. mobaraensis.1. It is also interesting to note that the gene for SvSS does not seem to be expressed under laboratory culture conditions, as the analysis of volatiles released by S. violarus did not show the presence of 1 or its autoxidation products 8 and 9. Many terpenoids have been isolated from bacteria to date,In summary, we have identified and mechanistically characterised the second bacterial sesterterpene synthase SvSS for sesterviolenes from 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.Supporting InformationClick here for additional data file.


Further extensive anion binding investigations with the most potent Sb‐ and Bi‐based PnB receptors: 2CF31⋅Sb and 2CF31⋅Bi, reveal novel selectivity profiles, both displaying Cl− selectivity relative to the heavier halides and, impressively, to a range of highly basic oxoanions. The synthesis and preliminary chloride anion binding studies of a series of novel tripodal tris‐proto‐triazole triaryl Sb(III) and Bi(III) mixed PnB‐HB receptor systems are also described. Whereas parent triphenyl Sb(III) and Bi(III) compounds are incapable of binding Cl− in THF solvent media, the PnB‐triazole HB host systems exhibit notable halide affinity.The synthesis and characterisation of a library of acyclic antimony(III) and bismuth(III) triaryl pnictogen bonding (PnB) receptor systems are reported. In the first‐generation receptor series, quantitative  A series of Antimony(III) and Bismuth(III) pnictogen bonding (PnB) triaryl‐based anion receptors demonstrate PnB anion binding strength is influenced strongly by the electronic withdrawing nature of the aryl substituent and pnictogen atom donor identity. The triaryl PnB receptors display chloride anion selectivity over a range of halides and oxoanions. The synthesis and preliminary chloride anion binding investigations of the first mixed PnB‐HB anion host systems are also described. In recent years sigma‐(σ)‐hole interactions, broadly defined as the attractive non‐covalent interactions between an electrophilic region of a main‐group element and a nucleophilic Lewis base, have been the subject of intense interest from the supramolecular chemistry community.3, 4 HR1⋅Sb and R1⋅Bi  and bismuth(III) triaryl acyclic receptor systems. In the first receptor series, 3 or BiCl3 afforded the target triaryl receptors, in variable yields, after purification by silica gel column chromatography. In the case of the cyano‐ and nitro‐appended triaryl pnictogens: CN1⋅Sb, CN1⋅Bi, NO21⋅Sb and NO21⋅Bi synthesis of the requisite organometallic precursor was achieved by a metal‐halogen exchange reaction between the appropriately functionalised aryl iodide and phenyllithium or isopropylmagnesium bromide. The consistently low yields for the cyano‐ and nitro‐triaryl pnictogens, for example 3 % for CN1⋅Sb, may be rationalised by undesired side reactions of organometallic reagents and the cyano or nitro groups.1H, 13C NMR, CHN elemental analysis and where possible single crystal X‐ray structural analysis, representative examples are shown in Figure In general, the first and second generation PnB receptor series were synthesised via treatment of the corresponding Grignard or organolithium reagent to a suspension of either antimony(III) or bismuth(III) trichloride, as summarised in Scheme 1, which in turn was prepared from a chemoselective Sonogashira reaction between 1‐bromo‐3‐iodobenzene and TMS acetylene,3 or BiCl3 to yield the tris‐TMS‐alkynes 2 and 3 in yields of 10 % and 15 % respectively. TMS deprotection was achieved through base promoted hydrolysis with K2CO3 in MeOH:CHCl3 mixtures to give the target proto‐alkynes 4 and 5 in excellent yield. The obtained tris‐alkynes were used in subsequent copper(I) catalysed azide–alkyne cycloaddition reactions with 3 equivalents of either benzyl or perfluorophenyl azide in the presence of catalytic TBTA and [Cu(MeCN)4]PF6 in anhydrous dichloromethane under an atmosphere of nitrogen. After stirring at room temperature for approximately 16 h, TLC analysis of the crude reaction mixtures indicated complete consumption of the alkyne starting materials and the formation of new species of considerably higher polarity. Subsequent NH4OH/EDTA(aq) work‐up procedures and purification by column chromatography gave the four novel tris‐proto‐triazole containing Sb(III) and Bi(III) receptors, Bz2⋅Sb, Bz2⋅Bi, PFP2⋅Sb and PFP2⋅Bi, in yields in the range of 14–61 % and were characterised by 1H, 13C NMR spectroscopy, CHN elemental analysis and high resolution ESI‐MS. In addition, crystals of PFP2⋅Sb and PFP2⋅Bi suitable for X‐ray diffraction analysis were obtained from slow vapour diffusion of pentane into chloroform solutions of the tripodal receptors  salt, to solutions of the receptors induced significant perturbations of various proton resonances. Typically, the ortho‐substituted proton environment to the pnictogen centre exhibited the largest perturbations, consistent with an anion recognition event mediated by PnB⋅⋅⋅Cl− interactions  values reveals several noteworthy trends. Firstly, with increasing electron withdrawing capability of the aryl substituents, reflected in the larger positive value of the aryl group's summed Hammett substituent constants (Σσ), there is a general increase in chloride affinity, consistent with an inductive activation of PnB donor atom electrophilicity, culminating in the largest association constants observed for the bis‐trifluoromethyl functionalised receptors 2CF31⋅Sb and 2CF31⋅Bi with aK(Cl−) values of 702 M−1 and 1,300 M−1 respectively. Furthermore, the bismuth triaryl receptors exhibit consistently enhanced chloride affinities relative to their antimony congeners, which is concordant with the accepted trend of increasing σ‐hole donor potency progressing down a given main group. To quantitatively evaluate the effect of changing the pnictogen atom donor identity in these receptor systems, chloride anion binding enhancement factors (α=KaBi/KaSb) were determined, facilitating comparison of the relative augmentation of association constant magnitudes. Interestingly, with the exception of the dichloro‐substituted aryl derivative,In order to assess the anion binding properties of the s Figure a and b. s Figure c, obtain1H NMR titration experiments with a range of anions including the remaining halides and a series of oxoanions were undertaken with the PnB receptors exhibiting the strongest chloride affinities, 2CF31⋅Sb and 2CF31⋅Bi. In all cases, similar downfield chemical shift perturbations were observed upon the addition of the anionic guests  centre's anion binding capabilities by appending electron‐withdrawing aryl units, we sought to further exploit the triaryl pnictogen framework not only as a Lewis acidic PnB donor, but also as a functional tripodal scaffold which could be further elaborated with additional cooperative triazole HB donor anion binding sites as in  Figure a. In ords Figure b.aK(Cl−) values for the tripodal receptor series reveals that the higher electrophilicity of Bi relative to Sb results in larger chloride affinities, by approximately a factor of two. Furthermore, the tripodal receptor series exhibits markedly enhanced chloride affinities relative to the corresponding parent triphenyl antimony (SbPh3) and bismuth (BiPh3) compounds, which exhibit no measurable chloride anion affinity.− binding exhibited by the tripods are indeed as a result of concerted and cooperative PnB and HB formation to the halide anionic guest.PFP2⋅Sb and PFP2⋅Bi outperform their benzyl functionalised analogues Bz2⋅Sb and Bz2⋅Bi. This is concordant with HB donor abilities typically correlating with C−H acidity, which is anticipated to be enhanced by the strongly electron deficient perfluoroaryl substituents.Bindfit analysis of the titration data determined 1 : 1 host:guest stoichiometric association constants for the tripodal series which are summarised in Table R1⋅Sb and R1⋅Bi, it is demonstrated that the bismuth analogues consistently outperform their antimony counterparts in terms of anion affinity. Importantly the electrophilicity of the PnB donor is demonstrated to be highly tunable and sensitive to the electronic‐withdrawing nature of the appended aryl groups. Furthermore, the two most potent Sb‐ and Bi‐based PnB receptors: 2CF31⋅Sb and 2CF31⋅Bi, exhibit Cl− selectivity relative to the heavier halides and, impressively, to a range of highly basic oxoanions, which is in stark contrast to anion selectivity profiles typically exhibited by traditional acyclic HB donor systems. We also report the synthesis and preliminary chloride anion binding investigations of a series of tripodal tris‐proto‐triazole triaryl pnictogen receptors as the first examples of a mixed PnB‐HB anion host system, wherein the Sb(III) or Bi(III) centre serves not only as a structural component providing a C3V scaffold for C−H HB donor arrays, but also as a functional Lewis acidic PnB donor. In comparison to the corresponding parent triphenyl antimony (SbPh3) and bismuth (BiPh3) compounds, which are unable to bind Cl− in deuterated THF solvent media, the mixed PnB‐triazole HB host systems display notable halide affinity. Importantly, all of these findings not only demonstrate the electronic substituent tunability of PnB donor anion binding potency, but also the potential for PnB integration into structural multifaceted anion host framework design, the subject of which is continuing in our laboratories.In summary, a library of acyclic Sb(III) and Bi(III) based triaryl PnB receptors have been prepared and extensive anion recognition studies undertaken. In the first receptor series General information as well as further details about compound synthesis, characterisation and X‐ray diffraction data are detailed in the Supporting Information.1⋅Sb2Cl), 2176087 (for 1⋅Sb3Cl), 2176083 (for 1⋅Sb2CF3), 2176085 (for 1⋅Bi2Cl), 2176088 (for 1⋅Bi3Cl), 2176082 (for 1⋅Bi2CF3), 2176089 (for 2⋅SbPFP) and 2176086 (for 2⋅BiPFP)2176084 (for  contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition Number(s) The authors declare no conflict of interest.1As 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.Supporting InformationClick here for additional data file.


Thus, we completed the total syntheses of (+)‐mycaperoxides D, C, G methyl ester, and (−)‐mycaperoxide B in 11 to 15 steps, confirming their original assignment.Mycaperoxides are natural endoperoxides isolated from different Mycale genus sponges, showing significant antiviral or antibacterial activities. We report herein the first total syntheses of representative congeners of this family from sclareol using a stereodivergent approach. Thus, an innovative oxidative ring expansion of cyclobutanol was used to bring the 1,2‐dioxane subunit, and a Mukaiyama aldol reaction on peroxycarbenium species was utilized to install the propionic acid subunit. During the study toward (+)‐mycaperoxide D methyl ester ‐Mycaperoxides D, C, G methyl ester, and (−)‐mycaperoxide B were synthesized in 11 to 15 steps for the first time. A stereodivergent approach from sclareol was adopted, highlighting ring expansion of strain cyclobutanol into 1,2‐dioxane, and Mukaiyama aldol reaction on peroxycarbenium species to install the propionic acid subunit. Prianos, Sigmosceptrella, Lacuntrulia, Diacarnus or Mycale. Structurally, these endoperoxides have a common backbone consisting of a 1,2‐dioxane ring substituted by a propionic acid subunit on one side and a sesquiterpene subunit connected via a methylene bridge on the other side. The main difference between all members lies in the nature of the sesquiterpene side chain and, in particular, the degree of cyclization of the chain, the intervention of methyl transpositions, and the enantiomeric series of this chain. The absolute configuration of the stereogenic centers of this subclass of natural products is not identical from one product to another, depending mainly on their origin. Despite many members, this subclass has been barely studied at the synthetic level. Indeed, Harwood reported several synthetic studies towards the total synthesis of mycaperoxide B, leading, with difficulty, to the obtention of four inseparable diastereomers, in a total of 16 steps, sharing the gross structure of the target. However, none of them corresponded to the natural product. was thus performed by silica gel chromatography. Therefore, three fractions  were obtained in 8 %, 32 %, and 31 % yield, respectively; each containing a mixture of defined diastereomers among the eight possible , a further separation of diastereomers was again done to deliver thioesters 16 a–h. All cis‐1,2‐dioxanes could be isolated as single stereoisomers but, trans‐1,2‐dioxanes, do not allow a separation of their 2,3,6‐epimer and were thus obtained as a mixture of two diastereomers. For the trans‐1,2‐dioxanes mixtures, a selectivity about 55 : 45 in favor of one particular 2,3,6‐isomer, allowed the differentiation of carbon for attribution.The synthesis of 13C NMR chemical shift. Firstly, cis/trans‐1,2‐dioxanes were assigned thanks to 13C NMR and the characteristic chemical shifts of the methyl groups at position C6 (20.0–21.0 ppm for trans‐dioxanes vs. 23.5–23.7 ppm for cis‐dioxanes) and of position C7 (43.0–43.9 ppm for trans‐dioxanes vs. 37.7–39.1 ppm for cis‐dioxanes). Secondly, the 2,3‐syn or 2,3‐anti configurations were then attributed based on the characteristic chemical shifts of the methyl groups at position C2  and of position C4 . Thirdly, 2,3,6‐epimer pairs  were the most difficult to discriminate due to the slight chemical shift differences due to the spacer between decalin and 1,2‐dioxane units. Structure of cis‐dioxanes 16 g and 16 h were deduced from the similarities observed on 13C NMR with natural (−)‐mycaperoxide D. Although tight chemical shift differences at C7 and C8, these positions are characteristic  and cis‐dioxanes 16 c and 16 d were next assigned. A more significant difference can be observed at C11 for cis‐dioxanes 16 c and 16 g versus trans‐dioxanes 16 d and 16 h (+1.6 ppm), which is indicative of a hydrogen bond of the hydroxyl at C10. Indeed, DFT calculation at the B3LYP/6‐31G showed that among the lowest energy conformers, an evident hydrogen bond exists between this hydroxyl group and carbonyl group at C1 with a distance of 2.18 Å ; lit:c 0.3, CHCl3)] confirmed the original assignment of mycaperoxide D (Scheme Among the eight diastereomers ‐mycaperoxide C methyl ester (1) was investigated. Thus, endoperoxyacetal 19 was obtained in five steps from 10 b, using a sequence of ring‐expansion of cyclobutanone 17 producing endoperoxide 18, similar to those described for the total synthesis of 2. A notable difference between the two synthetic routes lies in using TMS as the protecting group for the hydroxy group at C10. The conformation of 10 b did not allow the introduction of TES; consequently, only the use of TMS‐triflate with Et3N succeeded in the protection step. However, the use of TMS led to an unexpected consequence. Upon Mukaiyama‐type aldol reaction, in the presence of Sc(OTf)3 on 19, the hydroxyl at C10 was eliminated. The use of less active Y(OTf)36 and C2 to that was obtained in the total synthesis of 2. Again, during this synthesis, the formation of the cis‐dioxane rings (C3) was favored over the trans one, with a 2 : 1 cis:trans ratio. As described in the total synthesis of 2, we obtained a mixture of diastereomers distributed over three fractions. The fraction F3 was also containing the isomer corresponding to the natural product according to 13C NMR data. After deprotection of the alcohol function, the compound of interest 21 h was separated from its isomer 21 g. The final transesterification performed on 21 h led to the synthesis of the (−)‐mycaperoxide methyl ester C (1). Spectral data matched to the described natural product and, again, the optical rotation  confirmed the synthesis of ent‐mycaperoxide C methyl ester and the original assignment of mycaperoxide C 3) and (−)‐mycaperoxide B (4), their total synthesis was planned from compound 10 a. The same reaction sequence used to prepare intermediates 13 and 19 3 was used again, which led to the obtention of two fractions, F1 and F2 in 87 % yield . The fraction of interest, F2, contained a mixture of three products, including isomers 25 e and 25 f, which corresponded to the relative stereochemistry of mycaperoxide B (4) and (+)‐mycaperoxide G methyl ester (3) respectively. The utilization of preparative HPLC for fraction F2 led to the separation of isomer 25 h25 e/25 f. Thioesters 25 e and 25 f were saponified, providing mycaperoxide B (4) in an inseparable mixture with 26 e isomer in about a 53 : 47 ratio. Assignment of isomers 25 e and 25 f (or 26 e and 4) was tricky due to tenuous differences in the 13C NMR chemical shifts for those compounds [δ=0.1 ppm only for three carbons ]. Data for synthetic 4 were slightly closer to the reported natural mycaperoxides B than isomer 26 e, thus seeming to confirm the original statement. (Scheme Regarding (+)‐mycaperoxide methyl ester G  and (−)‐25 e–g, elimination of hydroxyl group induced by the action of SOCl2 led selectively to the tetrasubstituted olefin. Compound 27 g and a mixture of 27 e and 27 f were thus obtained. The combination of 27 e/27 f was transesterified, finalizing the synthesis of the (+)‐mycaperoxide methyl ester G (3) as a mixture with 28 e. As for 26 e and 4, the 13C NMR data for products 3 and 28 e were very close. The most significant differences were observed for the ethylenic carbons at C9 and C10 [δ=0.2 ppm]. The synthetic product with NMR signals matching the most with the natural product was attributed to structure 3 (Scheme From the fraction F2 containing isomers  ester G  as a mix1) and D (2) were obtained in 15 and 11 steps, respectively. The methyl ester of (+)‐mycaperoxide G (3) and (−)‐mycaperoxide B (4) were obtained in 14 and 13 steps but combined with their respective 2,3,6‐epimer. Original assignments were thus confirmed. We highlighted the utility of yttrium compared to scandium in the Lewis acid‐catalyzed Mukaiyama aldol reaction, which allowed smoother conditions reducing elimination side reaction with tertiary alkoxides. Although our approach did not allow us to control all the stereogenic centers of the synthetic targets, the different mycaperoxides studied were obtained efficiently accordingly to literature precedents.[syn/2,3‐anti and cis/trans‐1,2‐dioxanes relationships are easily determined by this method. However, the assignment of the 2,3,6‐epimers is more challenging, leading to small differences in NMR spectral data. Moreover, the affected positions are also a function of the nature of the decalin unit, which makes a general rule impossible for these pairs of diastereomers. This project also underlines the lack of enantiomeric methods to build organic peroxides, with few reported examples.[In summary, we were able to construct for the first time several members of the mycaperoxides, about thirty years after their discovery, employing an oxidative expansion of cyclobutanol to 1,2‐dioxane followed by a Mukaiyama aldol reaction on a peroxycarbenium species as key steps. Thus, the methyl esters of (+)‐mycaperoxides C  should be addressed to the authors.Supporting InformationClick here for additional data file.


Following the publication of this article , concernThe Fig 6d panel appears to partially overlap with Fig 9 panel G7 of  when rotFig 7a of this article  appears ○ Fig 7G of ○ Fig 4e of *○ Fig 8C of ○ Fig 4e of ○ Fig 5F of ○ Fig 5 panel G3 of Fig 7e of this article  appears ○ Fig 5a of ○ Fig 6 panel G1 of ○ Fig 10A of ○ Fig 6D of *Fig 8a panel of this article  appears PLOS ONE Editors are concerned about the reliability of data management and/or reporting for this study [The authors did not provide a response to the concerns raised with this article and did not provide the data underlying this study for editorial review. Given the nature and extent of the issues, the is study .PLOS ONE Editors retract this article.In light of the above concerns, the Some figure panels discussed above appear to report previously published material that are offered under a CC BY license, but the original articles were not attributed in . For thePH, AN, and MAA agreed with the retraction. MH responded but expressed neither agreement nor disagreement with the editorial decision. MFH, RMS, DAB, NSAW, and AA either did not respond directly or could not be reached. MAA stands by the article’s findings.