A wide range of structurally diverse polyheterocyclic compounds containing spirocyclic-fused butyrolactone-pyrrolidine-indoline and butyrolactone–pyrrolidine–dihydrobenzofuran skeletons could be smoothly obtained with excellent results (>99:1 dr and 98% ee). The potential synthetic applications of this methodology were also demonstrated by the scale-up experiment and by the diverse transformations of one product. This method is characterized by high asymmetric induction, wide functional group tolerance and scalability, and attractive product diversification.Catalytic asymmetric dearomative [3+2] cycloaddition of  A exhibits significant anticancer activity  cycloaddition, affording major diastereomers products 3d–h in 42–65% yields with acceptable diastereoselectivities and excellent ee values (92–96% ee). However, for the reaction between 5-OMe substituted 3-nitroindole with α-imino γ-lactone 2a, only a trace amount of product could be observed. On the other hand, the investigation of the α-imino γ-lactones was also conducted by using the standard reaction conditions. Both electron-donating and electron-withdrawing substituents at different positions on the phenyl ring of α-imino γ-lactones were well tolerated in reaction with 3-nitroindole 1a, providing the desired cycloaddition products 3i–l in 38–61% yields with good diastereoselectivities and 78–90% ee for their major diastereomers. Moreover, the disubstituted substrate also could smoothly react with 3-nitroindole 1a to give the corresponding product 3m in 48% yield for the major diastereomer with 59:15:26:0 dr and 80% ee. As for bulker naphthyl-substituted α-imino γ-lactone, the developed catalytic system also showed good compatibility, furnishing the product 3n in 67% yield with good stereoselectivity. Additionally, the heteroaromatic substrate, such as 3-fury-substituted α-imino γ-lactone, also efficiently worked in this asymmetric dearomative [3+2] cycloaddition reaction, providing the desired product 3o in 40% yield for the major diastereomer with 54:12:34:0 dr and 91% ee. To our disappointment, the α-imino γ-lactones 2 with strong EDG (-OMe) or EWG groups (–NO2 and –CF3) in the phenyl ring by reacting with 3-nitroindole 1 became messy or showed very poor reactivity, and no expected products could be obtained. The absolute and relative configuration of product 3a was unambiguously determined to be C7R, C8R, C12S, and C16R by single crystal X-ray analysis, the configurations of all other products in With the optimized reaction conditions in hand, we set out to investigate the substrate scope and generality of the copper-catalyzed asymmetric dearomative [3+2] cycloaddition reaction of different 3-nitroindoles 1 and α-imino γ-lactones 2, we next attempted to apply the similar catalytic system to the asymmetric dearomative [3+2] cycloaddition reaction between α-imino γ-lactones 2 and 2-nitrobenzofuran substrates 4 -Ph-PHOX ligand L4 as the catalyst, Cs2CO3 as the base, the asymmetric dearomative [3+2] cycloaddition reaction of 2-nitrobenzofuran 4a and α-imino γ-lactone 2a smoothly proceeded in PhCl at 30 °C, furnishing the desired product 5a in 61% yield for the major diastereomer with 76:24:0:0 dr and 96% ee. As for the 2-nitrobenzofurans bearing various electron-withdrawing groups such as F-, Cl-, and Br- on the aryl ring, these substrates could work well in reaction with α-imino γ-lactone 2a under the standard conditions to give products 5b–d in 80–86% yields with excellent stereoselectivities (>99:1:0:0 dr and 94–98% ee). Likewise, 2-nitrobenzofurans bearing an electron-donating group were also viable for the developed protocol, as demonstrated in the synthesis of products 5e and 5f in good yields with 97% ee and 93% ee, respectively. On the other hand, the asymmetric dearomative [3+2] cycloaddition reactions of diverse α-imino γ-lactones containing either electron-withdrawing or electron-donating groups on the aryl ring with 2-nitrobenzofuran 4a could smoothly occur under the standard conditions, delivering the corresponding dearomative cycloaddition products 5g–j in 53–85% yields with good stereoselectivities for their major diastereomers (up to 98:2:0:0 dr and 90% ee). In addition, the α-imino γ-lactone with a double substitution pattern could also smoothly react with 2-nitrobenzofuran 4a under the standard conditions to generate product 5k in 51% yield for the major diastereomer with 88:12:0:0 and 70% ee. The bulky naphthyl group was also tolerated well with the catalytic system, resulting in the formation of product 5l in moderate yield with excellent diastereoselectivity and good enantioselectivity. Moreover, the 3-fury substituted substrate could smoothly react with 2-nitrobenzofuran 4a, delivering the corresponding product 5m in moderate yield with 66:34:0:0 dr and 91% ee. However, when para-OMe- or ortho-NO2-substituted α-imino γ-lactone was tested, no expected products could be obtained. The absolute and relative configuration of product 5c was determined to be C5R, C6R, C7R, and C8R by single crystal X-ray analysis, the configurations of all other products in Having established a general scope to the catalytic asymmetric dearomative [3+2] cycloaddition reaction between 3-nitroindoles 1a and α-imino γ-lactone 2a was conducted under the standard conditions. As shown in 3a in 94% yield for the sum of the diastereomers with 51:20:29:0 dr and 96% ee. To our delight, the major diastereomer of product 3a could be easily isolated by flash column chromatography on silica gel  without any loss of the enantioselectivity (>99:1 dr and 96% ee). Moreover, different synthetic transformations of 3a to other heterocyclic compounds were also performed . In addition, 3a was subjected to tributyltin hydrogen and AIBN in toluene at 150 °C for 96 h in a sealed tube, thus affording compound 8 in 77% yield with >99:1 dr and 91% ee via a radical denitration process. Ultimately, the bromination reaction of 3a with N-bromosuccinimide (NBS) in CH3CN solvent could smoothly occur at 30 °C to produce compound 9 in 93% yield with >99:1 dr and 92% ee. In order to demonstrate the potential synthetic application of the developed catalytic asymmetric dearomative [3+2] cycloaddition reaction, a scale-up experiment between 3-nitroindole erformed . Treatinα-imino γ-lactones with electron-deficient nitroheteroarenes  with the solvent resonance employed as the internal standard (CDCl3 at 7.26 ppm and DMSO-d6 at 2.50 ppm). Data are reported as follows: chemical shift, multiplicity , coupling constants (Hz), and integration. 13C NMR chemical shifts are reported in ppm from tetramethylsilane (TMS) with the solvent resonance as the internal standard (CDCl3 at 77.20 ppm and DMSO-d6 at 39.52 ppm). The enantiomeric excesses were determined by chiral high-performance liquid chromatography (HPLC) analysis. High-performance liquid chromatography analysis (HPLC) was performed on Shimadzu SCL-10AVP HPLC systems consisting of a LC-10AD pump and an SPD-10A detector, measured at 254 nm. High-resolution mass spectrometer (HRMS) was recorded on Bruker Q TOF. Optical rotations were measured with a Perkin-Elmer-341 polarimeter. Melting points were recorded on a Büchi Melting Point B-545.Reagents were purchased from commercial sources and were used as received unless mentioned otherwise. Reactions were monitored by thin-layer chromatography (TLC). 4 (30 mg), ligand L1 , and Cu(OTf)2  were added to a Schlenk tube containing a magnetic stir bar under an argon atmosphere. chlorobenzene (1.0 mL) was added to the tube and the mixture was stirred for 1.0 h at room temperature. Then, in the resulting solution, a mixture including α-imino γ-lactones , Cs2CO3 , 3-nitroindoles , and chlorobenzene (1.0 mL) was subsequently added under an argon atmosphere at −30 °C. After being stirred for the appropriate reaction time, the reaction mixture was quenched with water. The organic layer was extracted with dichloromethane three times. The collected organic layer was dried over anhydrous Na2SO4. After the removal of the solvent under reduced pressure, the diastereomeric ratio was determined by 1H NMR analysis. The resulting crude mixture was purified by silica gel column chromatography to afford cycloadducts 3. The enantiomeric excesses of product 3 were determined by chiral stationary phase HPLC using a Chiralpak AD-H, AS-H, IA, IB, and Chiralpak IC column. MgSOL4  and Cu(OTf)2  were added to a Schlenk tube containing a magnetic stir bar under an argon atmosphere. Chlorobenzene (1.0 mL) was added to the tube and the mixture was stirred for 1.0 h at room temperature. Then, in the resulting solution, a mixture including α-imino γ-lactones , Cs2CO3 , 2-nitrobenzofurans , and chlorobenzene (1.0 mL) were subsequently added under an argon atmosphere at 30 °C. After being stirred for the appropriate reaction time, the reaction mixture was quenched with water. The organic layer was extracted three times with dichloromethane. The collected organic layer was dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the resulting crude mixture was purified by silica gel column chromatography to afford cycloadducts 5. The diastereomeric ratio and the enantiomeric excess values of product 5 were determined by chiral stationary phase HPLC using a Chiralpak AD-H, AS-H, IA, IB, and Chiralpak IC column.Ligand 4 (375 mg), ligand L1 , and Cu(OTf)2 (10 mol%. 90.5 mg) were added to a Schlenk tube containing a magnetic stir bar under an argon atmosphere. chlorobenzene (12.5 mL) was added to the tube and the mixture was stirred for 1.0 h at room temperature. In the resulting solution, the mixture including α-imino γ-lactone 2a , Cs2CO3 , 3-nitroindole 1a , and chlorobenzene (12.5 mL) were subsequently added under an argon atmosphere at −30 °C. After being stirred for 120 h, the reaction mixture was quenched with water. The organic layer was extracted three times with dichloromethane. The collected organic layer was dried over anhydrous Na2SO4. After the removal of the solvent under reduced pressure, the diastereomeric ratio was determined as 51:20:29:0 dr by 1H NMR analysis. The resulting crude mixture was purified by silica gel column chromatography using petroleum ether/ethyl acetate (8:1) as the eluent to afford cycloadduct 3a for the sum of diastereomers .MgSO3a   and trimethylsilyl chloride  in 1.0 mL of methanol at 0 °C. After the mixture was stirred at 0 °C for 2 h, the suspension was filtered and washed with methanol two times (2 × 5.0 mL) and dichloromethane two times (2 × 5.0 mL), respectively. After the filtrate was washed with saturated NaHCO3 aqueous two times (2 × 15 mL), the organic phase was separated while the aqueous phase was extracted with dichloromethane three times (3 × 15 mL). The combined organic phase was washed with brine three times (3 × 15 mL) and then dried with anhydrous Na2SO4. Subsequently, the organic phase was concentrated under reduced pressure. The residue was subjected to column chromatography over silica gel using petroleum ether/ethyl acetate (4:1) as the eluent to give compound 6 as a white solid .Newly activated zinc powder  was slowly added into a solution of 4  was cautiously added into the solution of compound 3a   in the 1.0 mL of THF at 0 °C. After the mixture was stirred at 0 °C for 1.0 h, the resulting mixture was quenched with 10 mL of 0.1 mol/L NaOH solution and extracted with dichloromethane three times (3 × 15 mL). The organic phase was isolated and washed with brine three times (3 × 15 mL), and then dried with anhydrous Na2SO4. Subsequently, the organic phase was concentrated under reduced pressure. The residue was subjected to column chromatography over silica gel using petroleum ether/ethyl acetate (2:1) (about 200 mL) as eluent, and then using MeOH as an eluent to give compound 7 as a white solid .LiAlH3a   and AIBN (azobisisobutyronitrile)  in toluene (1.0 mL). Then, the reaction mixture in a sealed tube was stirred at 150 °C for 96 h. Subsequently, the reaction mixture was cooled to room temperature, the solution was concentrated under reduced pressure. The residue was subjected to column chromatography over silica gel using petroleum ether/ethyl acetate (7:1) as an eluent to give compound 8 as a white solid .Tributyltin hydride  was added to a solution of compound N-Bromosuccinimide)  was added into the solution of compound 3a   in the 1.0 mL of MeCN, and the reaction mixture was stirred at 30 °C for 24 h. Subsequently, the resulting mixture was quenched with 10 mL of saturated NH4Cl solution and extracted with dichloromethane three times (3 × 15 mL). The combined organic phase was washed with brine three times (3 × 15 mL) and then dried with anhydrous Na2SO4. Subsequently, the organic phase was concentrated under reduced pressure. The residue was subjected to column chromatography over silica gel using petroleum ether/ethyl acetate (6:1) as an eluent to give compound 9 as a white solid .NBS  under mild reaction conditions. Importantly, these innovative polyheterocyclic compounds would be potentially promising for high-throughout drug screening due to the intriguing fusion of three privileged butyrolactone, pyrrolidine, and indoline/dihydrobenzofuran subunits into one molecular architecture. The potential synthetic applications of the dearomative [3+2] cycloaddition reaction were also demonstrated by the scale-up experiment and the diverse transformations of one product to diverse heterocyclic compounds. More importantly, this developed protocol also shows distinct features including high asymmetric induction, broad functional group tolerance and scalability, and attractive product diversification. The application of the asymmetric dearomative cycloaddition reaction of electron-deficient nitroheteroarenes toward library synthesis and subsequent biological evaluation of its members is underway.In summary, we have successfully developed a copper-catalyzed asymmetric dearomative [3+2] cycloaddition reaction of


To gain a comprehensive understanding of the catalyst's structure, composition, and morphology, various characterization methods were employed. These included FT-IR analysis to examine chemical bonds, SEM and TEM imaging to visualize the catalyst's surface and internal structure, TGA to assess thermal stability, EDS for elemental composition analysis, and XRD to determine crystal structure. The FeCeOx@g-C3N4 nanocatalyst demonstrated remarkable efficacy in the one-pot synthesis of 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazole. Noteworthy features of this catalyst included high percentage yield, mild reaction conditions, short reaction time, and an efficient and straightforward procedure. Furthermore, the FeCeOx@g-C3N4 composite exhibited excellent recyclability and reusability. It could be recycled and reused up to four times without a significant decline in catalytic activity.Herein, we developed a novel composite called FeCeO The presence of a lone pair of nitrogen in the imidazole ring enables the formation of hydrogen bonding, which contributes to their metal-binding capability. This property has found applications in the pharmaceutical industry5. In recent years, imidazole compounds have garnered widespread attention due to their diverse range of properties and applications. These compounds exhibit antibiotic, anti-tumoral, pesticide, herbicide, anti-allergy, anti-viral, and other pharmacological activities7. Moreover, the imidazole structure is present in various drugs such as losartan, eprosartan, histidine, and histamine8.Imidazole derivatives have gained significant importance due to their biological activity in natural products, biology, intermediates, and pharmacologically active compounds10. These imidazole derivatives have found numerous applications and gained importance across various industries11. Considering the significant applications of imidazole derivatives, several synthetic protocols with high yield and efficiency have been documented in the literature12. However, some of these protocols utilize toxic and expensive catalysts that are less efficient. Consequently, they often involve harsh reaction conditions, long reaction times, and yield limitations15.Two important imidazole derivatives are 1,2,4,5-tetraphenylimidazole and 2,4,5-triphenylimidazole. The first imidazole compound was reported by Radzisewski, Japp, and Robinson in 1882. They achieved its synthesis by reacting 1,2-dialdehyde with ammonium chloride. Since then, several imidazoles have been reported, derived from 1,2-diketones, α-ketomonoximes, α-hydroxy ketones in combination with aldehydes and ammonium3N4) has garnered significant attention and found applications in various energy-related fields16. g-C3N4 primarily consists of carbon and nitrogen atoms and possesses desirable properties such as easy synthesis and functionalization, excellent physicochemical stability, wide bandgap, low toxicity, and cost-effectiveness. The unique properties of g-C3N4, such as its functionalization capabilities, physicochemical stability, and low cost, make it an attractive choice for supporting other catalysts or functional materials21. One key advantage of g-C3N4 is its covalent bond nature, which results in an inactive surface that reduces the interaction between hydrogen and oxygen24. This characteristic enhances its catalytic properties26. However, pure g-C3N4 has two main limitations: first, it absorbs only a small fraction of solar energy, primarily in the range of low bandgap wavelengths (below 460 nm). Second, the fast recombination of double electrons within the cavities of g-C3N4 leads to a decrease in photocatalytic activity30. To address these limitations, various approaches have been developed to enhance the catalytic activity of g-C3N4 and mitigate imperfections31. These approaches include doping g-C3N4 with transition metals35 and coupling it with metals41. These strategies aim to improve the absorption of a broader range of solar energy, reduce electron recombination, and enable the recovery and recycling of g-C3N4, thereby enhancing its overall performance as a catalyst44.Graphitic carbon nitride , (3 1 1), (4 0 0), (5 1 1) and (4 4 0) plane diffractions of Fe2O3. In addition to the observed reflection at (3 1 1), (2 2 0), (2 0 0), and (1 1 1) belongs to CeO2. According to the typical characteristic diffraction peaks of g-C3N4, two characteristic diffraction peaks can be found at 13.1° and 27.4°.The crystal structure of the composite was characterized using a powder X-ray diffractometer (XRD). As shown in Fig. x@g-C3N4 nanocomposite. The FT-IR analysis confirms the presence of FeCeOx on g-C3N4. In Fig. −1 is attributed to the Fe–O stretching mode, providing evidence for the presence of FeO in the nanocomposite. Additionally, the absorption bands at 740 cm−1 and 1416 cm−1 are assigned to the Ce–O stretching vibrations, further confirming the presence of CeO2. The absorption band at 804 cm−1 corresponds to the bending vibration of the s-triazine ring in g-C3N4, indicating the presence of g-C3N4 in the nanocomposite. The absorption bands in the range of 1200–1400 cm−1 are attributed to the C–N stretching vibration mode present in g-C3N4. Furthermore, the absorption band at 1637 cm−1 corresponds to the C=N stretching vibration mode, providing further evidence of the presence of g-C3N4 in the nanocomposite. The broad absorption band observed in the range of 2800–3500 cm−1 is indicative of the N–H stretching vibrations of amine groups in g-C3N4 and the O–H stretching vibrations of absorbed water from the environment.FT-IR spectroscopy was employed to investigate the bonding states of the FeCeOx@g-C3N4 nanocomposite  is a technique used to study the thermal behavior of a material as a function of temperature. It involves measuring the weight changes of a sample as it is heated or cooled under controlled conditions Fig. . The TGAx@g-C3N4 nanocomposite were analyzed by ICP-MS. The amount of analyzed Ce (0.71 wt.%) corresponded to Ce content (0.69 wt.%) based on EDX measurement.To validate the applicability of ICP-MS as alternatives to EDX analysis for the measurement of Ce in the composite, FeCeOx@g-C3N4, the catalytic performance of the composites was investigated for the synthesis of 1,2,4,5-tetra phenyl imidazole and 2,4,5-triphenyl imidazole. This study aimed to develop a cost-effective and easily accessible method for synthesizing these imidazole derivatives using readily available starting materials. The catalytic protocol demonstrated excellent selectivity and simplicity, allowing for the synthesis of various 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles. The procedure presented a sustainable and chemically efficient alternative, as it utilized inexpensive and readily available starting materials.After preparing and characterizing FeCeO4OAc were chosen as model substrates to prepare 1,2,4,5-tetrasubstituted imidazole. The goal was to identify the optimal reaction conditions by varying catalysts, solvents, and temperatures. Table x@g-C3N4 as the catalyst (entry 8). Further investigation was conducted to determine the optimal catalyst amount (entries 7–10). The results in Table x@g-C3N4 as the catalyst (entry 8) provided the highest yield for synthesizing 1,2,4,5-tetraphenyl imidazoles using benzaldehyde (0.5 mmol), benzil (0.5 mmol), aniline (0.5 mmol), and ammonium acetate (0.5 mmol).In the optimization study, benzaldehyde, benzil, aniline, and NH3N4 nanocomposite as the catalyst, ethanol as the solvent, and a reaction temperature of 60 °C.The optimization study further investigated the effect of temperature and solvent on the synthesis of 1,2,4,5-tetrasubstituted imidazole. Table The relationship between reaction time and percent yield in the model reaction was evaluated by collecting experimental data points at various reaction times. These data points were then used to plot a graph, typically referred to as Fig. After optimizing the reaction conditions for synthesizing 1,2,4,5-tetraphenyl imidazole, various benzaldehydes were utilized to prepare different imidazoles. The results of these reactions are presented in Table x@g-C3N4. Finally, different amounts of FeCeOx@g-C3N4 nanocomposite were used to determine their effects on the reaction in the presence of ethanol at 80 °C  to reduce system errors and evaluate the catalyst's reusability. In this case, 100 mg of the FeCeOx@g-C3N4 nanocomposite, TGA analysis and FTIR spectroscopy were performed on both the fresh and recycled catalyst. The results from TGA analysis and FTIR spectra showed no appreciable changes in the chemical structure of the recycled FeCeOx@g-C3N4 after five cycles, further indicating its stability and suitability for reuse  plays a crucial role in this transformation. The presence of cerium oxide in the FeCeOx@g-C3N4 nanocomposite likely enhances the catalytic activity and stability of the catalyst. Additionally, the FeCeOx@g-C3N4 nanocomposite exhibits a synergistic effect, leading to increased yields of the trisubstituted imidazole product  and Fe(III) on graphitic carbon nitride (g-C3N4). This iron-based nanocomposite, in combination with cerium functionality and the good surface area of g-C3N4, shows great potential for one-pot preparations of imidazole derivatives, resulting in good to excellent yields within short reaction times. The heterogeneous catalyst is easily separated and can be reused in subsequent reactions. This nanocomposite offers several advantages as a catalyst for imidazole synthesis, including its efficient performance, easy separation, and recyclability.In summary, the FeCeO8 Advance diffractometer. Fourier transfer infrared spectroscopy (FT-IR) in Shimadzu IR-460. 1H NMR spectra were recorded on a 500 MHz spectrometer and 13C NMR spectra on a 125 MHz NMR spectrometer, respectively, using CDCl3 or DMSO(D6) as a solvent; chemical shifts have been expressed in ppm downfield from TMS.Melting points were measured in open capillaries with the Buchi 535 melting-point apparatus. The reactions were monitored by thin-layer chromatography (TLC) with UV light as detecting agents. EDS spectra and Scanning Electron Microscope (SEM) images were prepared via the TESCAN Vega3 Model. Powder X-ray diffraction (XRD) analyses were given in a Bruker AXS-D3N4 was synthesized by directly heating melamine in air methods44. 10 g of melamine powder was placed in a covered 50 mL alumina crucible and then heated in a muffle furnace at a ramp rate of 5 °C/min and kept for three h at 550 °C in air. After cooling to room temperature, a light yellow powder was collected and stored for further use. The g-C3N4 nanosheets are prepared by thermal exfoliation in the air. In detail, 2 g of bulk g-C3N4 was put into an uncovered crucible for heat treatment at 550 °C for 3 h to obtain white powder.According to our previous paper, the bulk g-C3N4 (400 mg), which was synthesized in the previous step, was placed in a 200 mL Erlenmeyer and then dispersed in 100 mL of methanol/water (1:1) using sonication for 10 min at room temperature. Then, 40 mg CeCl3 and 40 mg FeCl3 was dispersed in methanol, and 20 mL NaOH (2 M) was added to the mixture and sonicated for 10 min. Henceforward, the mixture was stirred for 5 h at 60 °C, and the mixture was filtered and washed with 10 mL of methanol and dried overnight at 60 °C under vacuum to obtain FeCeOx@g-C3N4.At first, the g-Cx@g-C3N4 (20 mg) as catalyst and ethanol (1 mL) as a solvent were added in a 5 mL round-bottomed flask respectively. The reaction mixture was stirred with a stirrer at 60 °C for 2 h, and TLC monitored the progress of the reaction. After the reaction was completed, 20 mL of ethyl acetate was added, the catalyst was removed by centrifuge, and the catalyst was washed with ethyl acetate and reused for the subsequent reactions. The organic residue was recrystallized to obtain 1,2,4,5-tetraphenyl imidazole and derivatives as pure products.To a mixture of aniline (0.5 mmol), benzaldehyde (0.5 mmol), benzil (0.5 mmol) and ammonium acetate (0.5 mmol), FeCeOx@g-C3N4 (20 mg) as a catalyst, and ethanol (1 mL) as a solvent were placed in a 5 mL round-bottomed flask, respectively. The reaction mixture was stirred with a stirrer at 80 °C for 100 min, and TLC monitored the progress of the reaction. After the reaction was completed, 10 mL of ethyl acetate was added, and the catalyst was removed by centrifuge. Then, the catalyst was washed with hot ethanol and ethyl acetate and reused for the subsequent reactions. The ethanolic residue was recrystallized to obtain 2,4,5-triphenyl imidazole and derivatives as pure products.A mixture of benzaldehyde (0.5 mmol), benzil (0.5 mmol), ammonium acetate (1.5 mmol), FeCeOSupplementary Figures.


Escherichia coli and other bacteria. This synthetic medium can be used to observe and evaluate the physiological activity of microbes under minimal nutritional requirements and determine the limiting factor for the desired phenotype. Although one of the advantages using the M9 medium is that its composition can be modulated, it is difficult to control presence of trace components and impurities from the reagents for preparing this medium. Herein, we showed that trace ingredients present in the reagents used for M9 media preparation affect the bacterial physiological activities . Additionally, we systematically identified the trace ingredient that influenced phenotypic differences. Our results showed that the selection of reagents and accuracy during reagent preparation is important for experimental reproducibility in the field of bio-engineering and systems biology focused on the systematic and continuous development of biomolecular systems .In the field of applied microbiology, reproducibility and experimental variability are important factors that influence both basic research as well as process development for industrial applications. Experimental reproducibility and accuracy depend not only on culture conditions such as temperature and aeration but also on raw materials and procedures used for media preparation. The M9 minimal medium is one of the most common synthetic media for culturing  The selection and development of the culture media are important for the evaluation of microbial physiology and to obtain microbes of the desired phenotype. The M9 minimal medium is one of the most common synthetic media used to cultivate Escherichia coli and other bacteria7. This medium contains inorganic sources of nitrogen (NH4Cl), phosphate (KH2PO4 and Na2HPO4), and sulphate (MgSO4), along with some other additives such as thiamine and trace metals. Furthermore, a sole carbon source  is also added to the M9 medium. Since the M9 medium is a synthetic medium, it can be used to observe and evaluate the physiological activity of microbes under minimal nutritional conditions and determine the limiting factor for the desired phenotype. Previous studies on microbial production of chemicals and fuel have employed the M9 medium as the basal medium13. In synthetic biology, the M9 medium has been used to evaluate the dynamic characteristics of synthetic genetic circuits21. Furthermore, the M9 medium is suitable for performing stable isotope tracing experiments, such as 13C turnover analysis and 13C metabolic flux analysis (13C-MFA)25 since unexpected contamination of 12C metabolites from natural nutrients  can be prevented. Additionally, the M9 medium containing U-13C6 glucose as the sole carbon source can be used to prepare a stable isotope-labelled internal standards mixture (SILIS)28. SILIS is essential for chromatography-mass spectrometry-based quantitative metabolome analysis using the stable isotope dilution method (SIDM)31. These studies suggest that the M9 medium can be utilised in diverse fields of microbiology.Naturally occurring as well as recombinant microbes have been employed in bioengineering for several processes, such as fermented food production7, Helmholtz Zentrum München (https://www.helmholtz-muenchen.de/), SubtiWiki32, etc. One of the media components, disodium phosphate (Na2HPO4), is available in both anhydrous and hydrated forms, and it is up to the experimenter to decide which reagent to use. Since anhydrous and hydrated disodium phosphate have the same chemical properties once dissolved in water at the same concentration, either recipe theoretically yields the same M9 medium. However, some researchers have shown empirically that microbial growth changes when the reagents and/or water used to make the media are altered34.There are several different M9 recipes available in the Cold Spring Harbor ProtocolsE. coli cultured on M9 media prepared using disodium phosphate of different grades and purity. Hydrated and anhydrous disodium phosphate of different grades were purchased from different sources to prepare the M9 media. Bacterial growth was evaluated on these media using standard techniques. Our results suggest that the purity of reagents used for preparing the M9 media might influence the growth of E. coli.Herein, we compared the physiological activity of 2HPO4 anhydrous (EP grade), Na2HPO4 · 7H2O (GR grade), and Na2HPO4 · 12H2O (EP grade) were purchased from Nacalai Tesque Ltd. while Na2HPO4 · 2H2O  was purchased from Merck KGaA.Disodium phosphate was purchased from two different companies [Nacalai Tesque Ltd. and Merck KGaA ] in different forms and grades Table . Na2HPO4E. coli strain BW25113  was lowest, and 51% lower than that observed in the M9 type A media. Furthermore, the specific growth rate (μ) of bacterial cells was also different in different M9 media  in M9 type C was significantly lower than that in other M9 media  were the same. These media were then used to culture E. coli observed in different M9 media are not dependent on the phosphate concentration, but are influenced by differences in disodium phosphate. The reproducibility of this experiment was confirmed through three different experiments . After 24 h of cultivation, approximately 6% of the total media was evaporated   Fig. . ConsequE. coli growth in different M9 media. Our results showed that altering the culture temperature  affects the bacterial cell growth in different M9 media   at this temperature  on dia Fig. . E. coli°C) Fig. a–c. Cell°C) Fig. a, althouure Fig. g. Similaure Fig. i. Therefent Fig. , and aerent Fig. . This waent Fig. . IncreasE. coli substrains, including K-12, B, and BL21(DE3) strains  and B (1G)) and GR grade (A (GR) and B (GR)) were obtained. Since Na2HPO4 · 2H2O was available only with E. Merck KG, we used this reagent (B). For Na2HPO4 · 7H2O, we obtained one ACS Grade reagent (C (ACS)) from MP Biomedicals, Inc. As shown in Fig. E. coli was significantly different in M9 media prepared using different disodium phosphate reagents. Cell growth in M9 type C (ACS)  improved to the same extent as other M9 types  . All Na2HPO4 reagents used in this study were analysed, and five trace impurities were detected . As shown in Fig. max)    Fig. a. A posiax) Fig. d. Iron wts Table . The amoE. coli in M9 Type C with various concentrations of nickel  in a specific concentration range (0.06–3.7 μg/L)  was also increased (42–69%) under the same range of NiSO4  are oxidative enzymes that catalyse the reversible oxidation of molecular hydrogen to protons and electrons36. Hydrogenases play an important role in energy metabolism in several microorganisms37. Three types of [NiFe] hydrogenases have been identified in E. coli: Hyd-1, Hyd-2, and Hyd-3  and is an oxygen-tolerant H2 oxidiser. Hyd-2 is an O2-sensitive [NiFe] hydrogenase involved in anaerobic respiration and consists of subunits HybA, HybB, HybC, and HybO. Hyd-3 is a part of the formate hydrogenlyase complex (FHL) that is produced under fermentative conditions and is required for the disproportionation of formate to CO2 and H2.Ni is known to play an important role in the biology of some eubacteria. In d-3 Fig. a38. Hyd-OF1-ATPase under different non-neutral pH conditions41. Moreover, Hyd enzymes may function as intracellular pH monitors through FOF1-ATPase which interacts with the FHL complex composed of FDH and Hyd to regulate the cytoplasmic pH40. According to these reports, nickel-dependent hydrogenases can support the growth of E. coli under microaerobic and acidic conditions by facilitating the homeostasis of intracellular energy/redox and pH. To investigate the relationship between these [NiFe] hydrogenases and the enhanced growth of E. coli in the presence of nickel sulfate, we prepared E. coli strains by deleting genes responsible for the activity of [NiFe] hydrogenases . When these strains were cultured in M9 type C with 0.05 µg/L of NiSO4, no significant enhancement of cell growth was observed . Moreover, selection and preparation of culture media is important to systematically evaluate the effects of gene deletion or upregulation using common bioresource platforms such as bacterial strain libraries2HPO4, even when we intended to prepare the same medium. Furthermore, Ni-dependent hydrogenases assist the growth of E. coli on M9 minimal medium containing glucose as the sole carbon source and a very small amount of nickel as an impurity. Slight differences in medium preparation  should be considered when performing microbiological experiments to ensure consistency and reproducibility.Herein, we demonstrated that bacterial physiological activity is affected by impurities present in the reagents used for medium preparation exemplified with Na3BO3; 2860 mg/L, MnCl2 · 4H2O; 1810 mg/L, ZnSO4 · 7H2O; 222 mg/L, Na2MoO4 · 2H2O; 390 mg/L, CuSO4 · 5H2O; 79 mg/L, Co(NO3)2 · 6H2O; 49 mg/L) was purchased from Merk KGaA .All chemicals were purchased from Nacalai Tesque Ltd.  and Fujifilm Wako Pure Chemical Industry Ltd. , unless otherwise specified. The trace metal solution A5  used for each 5 × salts solution (Type A ~ D). The Each 5 × salts solution was used for preparation of M9 media (Type A ~ D) supplemented with 4 g/L glucose, 10 ppm thiamine hydrochloride, and 10 ppm trace metal solution A5.The 5 × M9 salts solutions were prepared using several different disodium phosphate reagents. Tables E. coli strains BW25113, K-12, B, and BL21(DE3) were obtained from National BioResource Project (NBRP-E.coli at NIG), Japan. All strains were stored in 15% glycerol stocks at − 80 °C. Single-gene knockout variants of BW25113, JW0955 (ΔhyaB), JW2962 (ΔhybC), and JW2691 (ΔhycE) were obtained from the Keio collection42 and used for multiple [NiFe] hydrogenase genes deletion via P1 transduction45.600) did not exceed the detection limit of the plate reader (OD600 ≦ 0.99) after 24 h of cultivation  was automatically measured every 10 min for 24 h.The glycerol stocks of each strain were inoculated into 5 mL of LB medium in 30-mL test tubes as the seed culture, which was then incubated at 37 °C with orbital shaking (250 rpm) overnight. The overnight grown seed culture was passaged into fresh M9 medium (0.01% v/v). Afterwards, 200 μL of this solution was dispensed into a 96-well plate, which was covered with a clear plastic lid without sealing to ensure adequate air supply. The covered plate was then incubated with orbital shaking in a multi-mode plate reader Synergy HTX . To prevent fogging of the plate lid due to medium evaporation, cultivation was carried out in the condensation control mode of the plate reader. The cultivation temperature and orbital shaking conditions were set to 37°°C and 269 rpm  unless otherwise specified. The initial inoculum amount for the 96-well main culture was set so that the final cell density  on LC-20AD, SIL-20ACHT, CTO-20AC, and RID-10A instruments  equipped with a ligand-exchange chromatography column . The samples were separated using an 85% acetonitrile aqueous solution. The column temperature was set to 60 °C and the flow rate was adjusted to 0.8 mL/min. Five microliter of each sample was injected.p-toluenesulfonate mobile phase. The column temperature was set at 40 °C and the flow rate was adjusted to 0.8 mL/min. CDD-10AVP was used as the detector for the post-column pH-buffered electrical conductivity. Post-column pH buffering was attained using a pH-buffering solution containing 5.0 mM p-toluenesulfonate, 20 mmol/L Bis–Tris, and 0.1 mmol/L EDTA. Five microliter of each sample was injected.Extracellular organic acids and phosphate were quantified using HPLC on LC-30AD, SIL-30AC, CTO-20AC, and CDD-10AVP instruments (Shimadzu Corp.) equipped with three tandem ion-exclusion chromatography columns  and a guard column (Shim-pack Fast-OA (G), 4.0 × 10 mm, 5 mm, Shimadzu Corp.). Sample separation was achieved using a 5.0 mM 2HPO4 anhydrous , Na2HPO4 · 2H2O , Na2HPO4 · 7H2O , and Na2HPO4 · 12H2O . Result was normalised to the weight of the disodium phosphate reagent used for media preparation.Aqueous solutions of disodium phosphate (100 mg per 150 mL) were prepared for ICP-MS analysis using the following reagents: Na3 ; Barium Standard Solution  was diluted with 1% (v/v) HNO3 ; Chromium Standard Solution  was diluted with 1% (v/v) HNO3　; Iron Standard Solution  was diluted with 1% (v/v) HNO3 ; and Zirconium Standard Solution  was diluted with 1% (v/v) HNO3 .A calibration curve for each target ion was prepared using the following standard solutions: Nickel Standard Solution  was diluted with 1% (v/v) HNOICPMS-2030 (Shimadzu Co.) was operated with the following conditions: RF frequency power, 1.20 kW; plasma gas flow, 9.0 L/min; auxiliary gas flow, 1.10 L/min; carrier gas 0.70 L/min; nebulizer, coaxial; sampling depth, 7.0 mm; cone material, copper; internal standard, automatic addition (T-piece); sample tube, black-black PVC ; internal standard tube, orange-blue PVC .t-test and a p-value less than 0.05 was considered statistically significant.All the statistical analyses were performed using Microsoft Excel 2016. Data are shown as individual data points with mean ± standard deviation. Sample size has been mentioned in each figure and/or figure legend. All experiments presented in the manuscript were independently repeated at least two times with consistent results. The statistical significance of each experiment was determined by the Welch’s two-tailed Supplementary Information 1.Supplementary Information 2.


Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.  •Stabilization of diverse Lassa virus glycoproteins enables their structural characterization•mAb 12.1F, belonging to the GP1-A cluster, inhibits matriglycan and LAMP-1 binding•GP1-A mAbs show glycan dependence with 19.7E demonstrating lineage-dependent binding•A trimer-preferring Ab S370.7 targets the GPC-B epitope Perrett et al. structurally characterize Lassa glycoproteins from four lineages  and determine the molecular interactions critical for GP1-A NAb engagement. They then use these stable glycoproteins to isolate an antibody from convalescent patient serum. Transmission more rarely occurs via nosocomial infection,,,,,The ongoing SARS-CoV-2 pandemic emphasizes the importance of pandemic preparedness for zoonotic pathogens, which—through climate and anthropogenic variables that increase the landscape suitability for zoonotic transmission—cause approximately 75% of infectious disease in humans.,,,,,,,,,,,The glycoprotein complex (GPC) is the only viral protein on the surface of LASV and presents the sole target for neutralizing antibodies (NAbs).,The largest anti-LASV Ab isolation study to date, which yielded 113 cloned human monoclonal Abs (mAbs) from memory B cells of LASV survivors, defines the canonical Ab competition groups: GP1-A, GPC-A, GPC-B, and GPC-C.,,,,,,,,in vivo protection, yet the full nanoparticle system complicates structural analysis.Previous structural work of a ligand-free, native-like GPC has been made difficult by the instability of the trimeric ectodomain,,Here, we utilize the I53-50A subunit as a scaffold to generate and characterize GPC trimers of LASV genotypes beyond the prototypical lineage IV strain Josiah. We focus on LASV lineages of public health concern including lineage II, one of the most common lineages that circulates widely in southern Nigeria; lineage V, which circulates in Mali and has decreased pathogenicity compared with lineage IV; and lineage VII, a newly described lineage isolated from a nosocomial infection in Togo with comparable pathogenicity to lineage IV.,,,As LASV has known antigenic differences that may affect humoral cross-reactivity,,,,LASV GPC has a highly dense glycan shieldWe next assessed whether the LASV lineages present GPCs with distinct structural features. Using single-particle cryo-EM, we optimized the conditions for freezing LIV, LII, LV, and LVII GPC-I53-50A trimers B. Becaus5VK27PVDThe GPC-I53-50A constructs recapitulate the known GPC structural features and domain organization.The main differences among the GPC lineages are found in flexible loops, most notably the loop (residues 166–181) extending from the β7 sheet prior to the α3 helix B. This o,2 upon 18.5C binding, for example, and lowers the solvent accessibility of the fusion peptide. Both the Ab-bound and unbound structures show the fusion peptides adopting a near-identical conformation starting at the fusion loop (residues 277–299).When comparing our structures with the Ab-bound structures reported previously,,,∗01 and IGKV3-11∗01, respectively, the germline HC and LC of 19.7E are IGHV3-74∗02 and IGKV1-5∗01. The VH genes of 12.1F HC and LC are 8.8% and 7.6% somatically hypermutated, respectively, based on the sequences publicly available (patent WIPO: WO2018106712A1).While GPC-A- and GPC-B-specific Ab interactions with GPC have been studied in detail,,To identify differences between 12.1F and 19.7E at the phenotypic level, we analyzed the GPC binding and neutralization of these mAbs to a broad panel of LASV lineages A. Using max values relative to 37.7H  of LIV GPC-I53-50A by >10°C. In contrast, 12.1F and 19.7E had only marginal effects on GPC thermostability, suggesting that GP1-A-specific mAbs likely do not neutralize by stabilizing the prefusion state of GPCs. To probe the interaction between the GPC and the matriglycan moieties of α-dystroglycan, we used a microarray presenting chemoenzymatically generated matriglycan oligosaccharides of a defined length.,5 and 0.5 × 105 without and with 12.1F, respectively, two-tailed Mann-Whitney U test; p = 0.029). In contrast, 19.7E showed 16% inhibition of matriglycan binding . Interestingly, the GPC-A-targeting mAb 25.10C also inhibited matriglycan binding while GPC-B mAb 37.7H did not . Our models reveal that both Abs bind near the apex of the trimer, with each Fab engaging a single GP1 subunit on the loop that extends over β5–β8. 12.1F uses both its HC and LC to interact with the GPC, while 19.7E almost exclusively relies on its HC. 12.1F and 19.7E both bind in the space between apical glycans N89, N109, and N167 and show extensive contacts with the GPC glycans with total buried surface areas of 1,549 and 1,123 Å2, respectively.To assess the molecular interactions between GP1-A-targeting Abs and GPC, we used single-particle cryo-EM and determined the structures of 12.1F and 19.7E bound to the LIV GPC A and 4B 2 (N167). Additional contacts are described in 7UOV; While our previous observations and 2D classifications suggest that 12.1F typically binds in a 2 Fab-per-1 GPC fashion, applying C3 symmetry to the data enabled the best resolution of the epitope-paratope interaction A. Amino 2 (N89). Upon GPC binding to 19.7E, the fusion peptide takes on a similar conformation as seen when complexed with GPC-A- and GPC-B-targeting NAbs and extends toward the trimer interior.,,For 19.7E, we typically saw one Fab bound per GPC trimer and thus used symmetry-expanded particles to achieve a subset of protomers bound to the Fab. This Ab makes more contacts with amino acid residues than 12.1F , entirelAs we noticed that the GP1-A-specific Abs shared extensive interaction networks with the apex glycans, we decided to assess whether neutralization by these mAbs is glycan dependent, as has been seen previously with the NAb LAVA01.,,Inspection of the structures support the LAMP-1 and matriglycan competition we observed for these GP1-A mAbs. The 12.1F and 19.7E Fabs come within close proximity of H92 , which—tOur ligand-free structures  enable m5 and 0.9 × 105 without and with S370.7, respectively, two-tailed Mann-Whitney U test; p = 0.029). Consistent with our inability to find a neutralization mechanism, S370.7 was unable to neutralize authentic Josiah virus  describe stable, trimeric GPC reagents for cross-binding assessment, (2) provide a robust and relatively high-throughput platform for single-particle cryo-EM analysis of LASV GPCs with and without mAbs, (3) inform more comprehensive immunogen design and stabilization work, specifically in the context of GP1-A-targeting Abs, and (4) allow for B cell sorting of strain-specific and broadly reactive mAbs.,While our GPC-I53-50A proteins share high structural similarity to full-length LASV GPCs A, it is We note that 12.1F inhibits matriglycan binding by 51% despite not overlapping with the known matriglycan binding site. At this time, the exact molecular mechanism by which these GP1-A-specific Abs affect receptor binding is unknown. Further studies are needed to define the exact mechanisms.andrew@scripps.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Andrew B. Ward , 1–424 (GenBank: AHC95553.1), and 1–423 (GenBank: AMR44577.1), respectively, with the GPCysR4 mutations introducedAAL13212.1), or lineage V (Soromba-R) was inserted by Gibson assembly. Q5 site-directed mutagenesis was used to introduce the S111A and N167Q mutations into a plasmid encoding full-length, native Josiah GPC (a kind gift from Robin Shattock).The LIV GPC monomer, LIV GPC-I53-50A, and Avi-his-tagged LIV GPC-I53-50A constructs were generated as described previously.6 cells/mL using PEImax at a ratio of 1:3 DNA to PEI. HEK 293F cells were maintained in HEK 293F in 293FreeStyle expression medium (Life Technologies) and cultured at 37°C with 8% CO2 while shaking at 125 rpm. Plasmids encoding GPCs were co-transfected with a furin plasmid at a 1:2 ratio. To express biotinylated GPC-I53-50A, HEK 293F cells were co-transfected with Avi-his-tagged GPC-I53-50A, furin and a BirA plasmid (a kind gift from Lars Hangartner) in a 2:1:0.5 ratio. IgG plasmids were transfected at a heavy and light chain ratio of 1:1 while the generation of Fabs of 12.1F, 25.10C, and S370.7 was initiated by transfecting the histidine-tagged heavy chain Fab domain with the corresponding light chain at a ratio of 1:2. Culture supernatants of GPC constructs were harvested after six days, while IgG and Fab were harvested after five days. GPC-I53-50As were purified by gravity column using StrepTactin 4Flow resin (IBA Life Sciences) according to manufacturer's protocol and eluted with 1X BXT (IBA Life Sciences). IgGs were purified by gravity column using Protein G (Cytiva) or CaptureSelect IgG-Fc resin (Thermo Scientific) and eluted with 0.1 M glycine at pH 2.0. Biotinylated GPC-I53-50As and Fabs of 12.1F and S370.7 were purified by rolling the culture supernatant overnight at 4°C with Ni-NTA Agarose resin (Thermo Scientific). The next day, the bead suspension was flown over a gravity column, washed with 20 mM imidazole, 50 mM NaCl, pH 7.0 and eluted with 500 mM imidazole, 50 mM NaCl buffer, pH 7.0. Recombinant LAMP-1 was generated by transfecting HEK 293F cells with a rabbit Fc-tagged LAMP-1 plasmid encoding residues A29-S351 (a kind gift from Thijn Brummelkamp).GPC-I53-50As, LIV GPC monomer, biotinylated GPC-I53-50As and Abs were transiently expressed in HEK 293F cells at a density of 1.0 x 10Tonset) and denaturation (Tm) temperatures using the Prometheus NT software.Thermostability of GPC and GPC-Fab complexes was determined with a nano-DSF NT.48 (Prometheus). GPC proteins or complexes were diluted to 0.5 mg/mL and loaded into high sensitivity capillaries. The assay was run with a linear scan rate of 1°C/min and 80%-100% excitation power. The first derivative of the ratio of tryptophan fluorescence wavelength emissions at 350 and 330 nM were analyzed to determine thermal onset . GPC-I53-50A samples were diluted in TBS to approximately 15 μg/mL and loaded onto the copper grids and incubated for 30 s. The sample was blotted and immediately stained with 2% (w/v) uranyl formate for 15 s. Excess stain was removed by blotting and grids were dried for >5 minutes before being loaded on a 200 kV Tecnai F20 electron microscope (FEI) featuring a TemCam F416 CMOS camera (TVIPS). Images were collected at a magnification of 62,000X with a defocus value of -1.5 um, total electron dose of 25 e100 μg aliquots of each sample were denatured for 1h in 50 mM Tris/HCl, pH 8.0 containing 6 M of urea and 5 mM dithiothreitol (DTT). Next, GPC-I53-50A samples were reduced and alkylated by adding 20 mM iodoacetamide (IAA) and incubated for 1h in the dark, followed by a 1h incubation with 20 mM DTT to eliminate residual IAA. The alkylated GPC-I53-50A samples were buffer exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (3 kDa) and two of the aliquots were digested separately overnight using chymotrypsin  or alpha lytic protease (New England Biolabs) at a ratio of 1:30 (w/w). The next day, the peptides were dried and extracted using C18 Zip-tip (MerckMilipore). The peptides were dried again, re-suspended in 0.1% formic acid and analyzed by nanoLC-ESI MS with an Ultimate 3000 HPLC (Thermo Fisher Scientific) system coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) using stepped higher energy collision-induced dissociation (HCD) fragmentation. Peptides were separated using an EasySpray PepMap RSLC C18 column (75 μm × 75 cm). A trapping column (PepMap 100 C18 3 μM 75 μM x 2cm) was used in line with the LC prior to separation with the analytical column. The LC conditions were as follows: 280 minute linear gradient consisting of 4-32% acetonitrile in 0.1% formic acid over 260 minutes followed by 20 minutes of alternating 76% acetonitrile in 0.1% formic acid and 4% ACN in 0.1% formic acid, used to ensure all the sample had eluted from the column. The flow rate was set to 200 nL/min. The spray voltage was set to 2.7 kV and the temperature of the heated capillary was set to 40°C. The ion transfer tube temperature was set to 275°C. The scan range was 375−1500 m/z. Stepped HCD collision energy was set to 15, 25 and 45% and the MS2 for each energy was combined. Precursor and fragment detection were performed using an Orbitrap at a resolution MS1=120,000. MS2=30,000. The AGC target for MS1 was set to standard and injection time set to auto which involves the system setting the two parameters to maximize sensitivity while maintaining cycle time. Full LC and MS methodology can be extracted from the appropriate raw file using XCalibur FreeStyle software or upon request.Glycopeptide fragmentation data were extracted from the raw file using Byos . The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified. The MS data was searched using the Protein Metrics 305 N-glycan library with sulfated glycans added manually. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted chromatographic areas for different glycotypes with an identical peptide sequence. All charge states for a single glycopeptide were summed. The precursor mass tolerance was set at 4 ppm and 10 ppm for fragments. A 1% false discovery rate (FDR) was applied. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted ion chromatographic areas for different glycopeptides with an identical peptide sequence. Glycans were categorized according to the composition detected.Purified GPC-I53-50A was incubated with purified Fabs for at least 1 h at 4°C at a 1:9 molar ratio of GPC-I53-50A to Fab. Next, complexes were purified from unbound Fab by size exclusion chromatography using a Superdex 200 increase 10/300 GL column. Fractions corresponding to GPC-Fab complexes (9-10.5 mL) were pooled and concentrated using a MWCO concentrator with a cutoff of 100 kDa (Millipore).2 plasma using a Solarus plasma cleaner (Gatan) for 10 s or were plasma discharged for 25 s at 15 mA using a PELCO easiGlow (Ted Pella Inc.). Right before applying the protein samples to the grids, we added flouro-octyl maltoside at a final concentration of 0.02% (w/v). Cryo-grids were prepared using a Vitrobot mark IV (Thermo Fisher Scientific). In all instances, the chamber temperature and humidity were set to 4°C and 100%, respectively. Samples were frozen using variable blot times between 3 to 7 s with a blot force of 1 s and a wait time of 10 s. After blotting, the grids were plunge-frozen in liquid ethane.To prepare grids for sample application, UltrAuFoil R1.2/1.3  grids were treated with Ar/O-/Å2. Magnifications were set to 130,000 or 36,000X for the Krios and Arctica, respectively. Automated data collection using the Leginon software packageCryo-grids were loaded into an FEI Titan Krios or Talos Arctica (Thermo Scientific), which operate at 300 or 200 kV, respectively. Both microscopes were equipped with a K2 Summit direct electron detector camera (Gatan). The data were collected with approximate cumulative exposure of 50 eImage preprocessing was performed using the Appion software package.7SGD) and I53-50A protein (PDB 6P6F). For GPC-I53-50A and Fab complexes, the ligand-free initial model was used for initial 3D refinement steps. After preliminary 3D maps were generated demonstrating Fab density, they were lowpass filtered and used as the initial model for subsequent steps.All datasets were analyzed using an initial model generated in UCSF ChimeraFor ligand-free GPC-I53-50As, preliminary 3D refinements were performed in cryoSPARC v3.0.For Ab-bound GPC-I53-50A structures, the same general processing steps were followed as above sans moving particles to Relion 3.1. LIV GPC-I53-50As bound to 12.1F and S370.7 were analyzed by imposing C3 symmetry after initial alignments. LIV GPC-I53-50A bound to 19.7E was analyzed by symmetry expanding the particle set after C1 alignment along the C3 axis of symmetry. Particles were sorted using focused classification using a 60 Å sphere mask around the epitope-paratope interface to distinguish particles with Fab density. Subsequent refinements were performed to constrain particle alignment to one protomer face.7SGD was used as the initial model and manually fit into density using Coot.,8EJH and 7UOVPost-processed maps were used to build all final atomic models. For LIV GPC-I53-50As, PDB Fabs of 19.7E were generated by papain digestion of purified IgG. First, a buffered aqueous suspension of papaya latex papain (Sigma Aldrich) was activated by incubating in 100 mM Tris, 2 mM EDTA, 10 mM L-cysteine at 37°C for 15 mins. Next, IgG was incubated with activated papain in 100 mM Tris, 2 mM EDTA, 10 mM L-cysteine at a ratio of 40 μg activated papain per 1 mg of purified IgG for 5 hours at 37°C. The reaction was quenched by adding iodoacetamide to a final concentration of 0.03 M. Undigested IgG and Fc fragments were removed by a 2 h incubation with CaptureSelect IgG-Fc resin (Thermo Fisher Scientific). Resin was spun down and the supernatant run on a Superdex 200 increase 10/300 GL column (Sigma-Aldrich) size exclusion column using TBS as its running buffer. Fractions from 15.5-16.5 mL elution volume were collected and concentrated in a MWCO concentrator (Millipore) with a 10 kDa cutoff.Ab binding to GPC-I53-50As was assessed using an Octet Red96 instrument (ForteBio). Biotinylated GPC-I53-50A was loaded onto SA sensors (Sartorius) at 100 nM. After a short dip in running buffer , sensors were dipped in IgGs diluted to 400, 200, 100, 50, 25, or 12.5 nM. For Fab measurements, the sensors were dipped in a 400 nM dilution of Fabs. Association and dissociation steps were measured for 600 s. Assays were performed at 30°C. All dilutions were made in running buffer with a final volume of 200 μL per well. Kinetics buffer references were subtracted from all data, which was then analyzed using Octet Data Analysis software. 12.1F, 19.7E, and S370.7 IgG kinetics were modeled assuming a 1:1 binding model while 37.7H assumed a 2:1 binding model because of its biphasic on-rate and poor fit with a 1:1 model.Biotinylated GPC-I53-50As diluted in running buffer  were loaded onto SA sensors (Sartorius) to a signal of 1.0 nm using an Octet Red96 system (ForteBio). After a short dip in running buffer, the sensors were dipped in 400 nM of 12.1F, 19.7E, S370.7, or 25.10C diluted in running buffer or running buffer alone. To measure IgG dissociation, the sensor was dipped for 1200 s in pH 5.0 running buffer . The sensor was then dipped for 600 s in 200 μg/mL of recombinant LAMP-1 ectodomain in pH 5.0 running buffer, after which the sensor was dipped in pH 5.0 running buffer for 1200 s to measure LAMP-1 dissociation.,50 values were determined as the concentration at which infectivity was inhibited by 50% using Prism 9 (GraphPad).LASV pseudoviruses were made as previously describedNeutralization assays using authentic LASV  were performed in the BSL-4 laboratory of the Institute of Virology, Philipps University Marburg, Germany as previously described.12.1F, 19.7E, 37.7H, and S370.7 IgGs were immobilized on AHC sensors (Sartorius) to a signal of 1.0 nM using an Octet Red96 instrument (ForteBio). The immobolized IgGs were then dipped in running buffer  followed by LIV GPC-I53-50A trimer, LIV GPC monomer, or running buffer. LIV GPC-I53-50A trimer and LIV GPC monomer were diluted in running buffer to concentrations that would contain the same amount of protomers in solution: 150 nM and 450 nM, respectively. Following a 600 s association period, the tips were dipped into running buffer and dissociation was measured for 600 s.The synthesis of matriglycan compounds were reported previously.Printed glass slide was pre-blocked with a solution of 1x TSM binding buffer  for 90 mins and the blocking solution was discarded. The Strep-tagged GPC-I53-50A featuring the native S1P cleavage site (1 μg/mL) was incubated with 12.1F, 19.7E, 25.10C, 37.7H, or S370.7 mAbs (5 μg/mL) in TSM binding buffer at 4°C for 1 h before StrepMAB-Classic Oyster 645 conjugate  was added, and the solution was further incubated for another 30 min at 4°C. For the detection of the mAb, a Cy3 conjugated goat-anti-human IgG Ab was used . The solution was then added to the microarray slide and the slide was incubated at room temperature for 1 h. The slide was sequentially washed with TSM wash buffer , TSM buffer  and water.https://doi.org/10.5281/zenodo.5146251) to provide the results. The highest and lowest value of the total fluorescence intensity of the six replicates spots were removed, and the four values in the middle were used to provide the mean value and standard deviation. Due to the small sample size normality of this data was not assumed. Instead, two-tailed Mann-Whitney U-tests were performed to compare matriglycan binding to GPC-I53-50A in different conditions. P value estimations using this test are capped at 0.029 based on the sample size.The slides were scanned using a GenePix 4000B microarray scanner (Molecular Devices) at the appropriate excitation wavelength with a resolution of 5 μM. Various gains and PMT values were employed in the scanning to ensure all the signals were within the linear range of the scanner’s detector and there was no saturation of signals. The image was analyzed using GenePix Pro 7 software . The data was analyzed with an Excel macro (We used two GPC bait constructs for isolating LASV-specific B cells: LIV GPC-I53-50A and Josiah rGPen), definition of center, dispersion measures (when applicable), and the statistical tests employed are noted in figure legends and/or results. Notably, for the synthetic matriglycan arrays, the small sample size insinuates the normality of the data cannot be assumed. Two-tailed Mann-Whitney U-tests were thus performed. P value estimations using this test are capped at 0.029 based on the sample size, and we consider P values <0.05 to be significant. All statistical analyses were performed using Graphpad Prism 8.0.The number of replicates (


Hole carriers were doped by substituting Zn with Cu until x = 0.02, resulting in an increase of the power factor from0.049 to 0.52 mW/mK2 at T = 797 K. Thelattice thermal conductivity was substantially low, with a value of1.61 W/mK at T = 312 K, independent of doping. Thiscan be attributed to the large vibration of the Rb atoms, as demonstratedby the neutron diffraction analysis. The maximum dimensionless figureof merit, ZT, was 0.53 at T = 797 K, representingthe highest value for the 143-Zintl compounds. The result indicatedthat the 143-Zintl compounds could be a new class of high-performancethermoelectric materials.The thermoelectricperformance of RbZn For example, Yb2Sb2 is a p-type compound exhibiting a ZT value of 1.2 at T = 700 K.11 Mg32 is a n-type compound exhibiting a ZT value of 1.65 at T = 725 K.14 There are also other Zintl compounds, for example,111-system.15 Characteristically, thesecompounds exhibit significantly low lattice thermal conductivities(κL), which facilitate high thermoelectric performance.Recently, we found that the 122-Zintl compounds of the ThCr2Si2-type structure could also be promising candidatesfor high-performance thermoelectric materials.16 A metastable state of Zn2As2 obtained by quenching from a high-temperature phase, exhibited aZT value of 0.3 at T = 773 K, with a substantiallylow κL value (<1 W/mK). The result motivated furtherexploration of the ThCr2Si2-type structure comprisinga large family of compounds.One successful system that has achieved the demand is 122-Zintlcompounds, having a chemical formula of AMA ion layer from every two MX layers  andRbCd4As3-type (R3̅m) structures derived from the ThCr2Si2-type and CaAl2Si2-type structures, respectively.22 Other reported structures for 143-Zintl compounds include CaFe4As3-type (Pnma), NaZn4Sb3-type (P63/mmc), and β-BaCu4S3-type (Cmcm) structures.24The 122-Zintl compounds can betransformed into various layeredcompounds by a combination of stacking layers. The 143-Zintl compoundsare among those that are derived by removing an X layers 1.17–24 T24 The RbCd4As3-type compounds of NaZn4As3 and NaCd4Sb3 exhibit ZT values of 0.067 and 0.044 at room temperature,respectively.21 Regarding the other types, NaZn4Sb3 (space group P21/c) exhibits ZT = 1.4 × 10–3 and KMg4Sb3 (β-BaCu4S3-type) exhibits ZT = 2.2 × 10–5 ata room temperature.24 Although all of them exhibitsufficiently low thermal conductivities, their ZT values were considerablylow because of their small power factors (S2/ρ).Recently, several research groups reported thethermoelectric propertiesof 143-Zintl compounds.4S3-type derived from the ThCr2Si2-type are alsoan attractive target; however, their thermoelectric property has notbeen reported. A first-principles calculation on RbZn4P3 (KCu4S3-type) yielded a high ZT valueof 0.78 at T = 700 K, supporting their value forexploring.25 Substituting P atoms withheavier As atoms can induce a lower lattice thermal conductivity,which leads to a higher thermoelectric performance. Thus, in thisstudy, we investigated the thermoelectric properties of Rb4As3 crystallized in the KCu4S3-type structure.143-Zintl compounds crystallized in KCu2x4–CuxAs3 were synthesizedby solid-state reactions using Zn (4N), Cu (3N), and As (6N), andpresynthesized RbAs was used as starting materials. RbAs was synthesizedusing Rb (3N) and As (6N) mixed at a stoichiometric composition. Theywere loaded onto an alumina crucible that was encapsulated in a screw-topstainless-steel container filled with an Ar gas26 and heated at 650 °C for 20 h. The obtained precursorwas mixed with other starting materials in a ratio of RbAs/Zn/Cu/As= 1:4.2-x:x:2 to synthesize RbZnx4–CuxAs3. The mixture was ground, pressed into a pellet, placed inan alumina crucible, and sealed in a screw-top stainless-steel containerfilled with Ar gas. The container was then heated at 800 °C for40 h. The mixing and grinding were performed in a glovebox filledwith dry Ar gas. Stainless-steel containers were heated in a box furnaceinstalled in a drafter for the precaution against leaking. Dense pelletswere obtained by subsequent hot pressing at 650 °C for 1 h undera uniaxial pressure of 70 MPa in an Ar gas flow. The relative densityof the obtained pellets was above 97%. Finally, the obtained pelletswere annealed at 500 °C for 10 h.Polycrystallinesamples of RbZnPowder X-ray diffractionanalysis was performed at room temperatureusing Cu Kα1 radiation that was generated at 40 kV and 45 mAwith a SmartLab (RIGAKU) diffractometer. The scan speed for the 2θaxis was 0.1°/min. The dense pellets were pulverized and loadedonto a glass capillary with an outside diameter of 0.4 mm for themeasurements.The electrical resistivity and Seebeck coefficientwere measuredin an Ar gas atmosphere using an RZ2001i (Ozawa Science) instrument.The electrical resistivity was measured by a four-probe method employingplatinum wires as the voltage probe. The Seebeck coefficient was measuredunder a temperature gradient employing Pt–PtRh thermocouplesas the temperature probes. The measurements were conducted along thedirection perpendicular to the uniaxial pressure applied to the hotpress.D) and specificheat(Cp) were measured by a laser flash method under an Ar gas flow. The measurements were conductedalong the direction perpendicular to the uniaxial pressure appliedin the hot press. The total thermal conductivity (κ) was calculatedusing κ = DCpds, where ds is the sample density.The values derived from the Dulong–Petit law were used forthe calculations, which were confirmed to be consistent with the experimentalvalues. The accuracy of the calculated Cp was also confirmed via differential scanning calorimetry using aDSC404 F3 (Netzsch) instrument under an Ar gas flow for a nondopedRbZn4As3 sample.The thermal diffusivity (RH) was measuredat room temperature by the van der Pauw method using a homemade instrument.A magnetic field was applied up to 2 T. The Hall carrier concentration(nH) was calculated from nH = 1/RHe, where e is the elementary charge. The Hall mobilitywas calculated by μH = 1/(nHeρ).The Hall coefficient reflection of a Ge monochromator. Thescattering angles (2θ) were moved at a constant step of 0.1°to cover the range of 3–153° using a multidetector. Thepowder samples were loaded in a vanadium cell and mounted in a cryofurnacefor measurements in the range of 106–663 K. The obtained diffractionpatterns were analyzed by the Rietveld method using RIETAN-FP to determinethe crystal structure parameters.4As3 using the WIEN2k package.28 The modified Becke-Johnson potential was assumed as theexchange–correlation potential,30 and spin–orbitcoupling was considered. The value of RKmax was set to 8 with 17 × 17 × 6 and 29 × 29 ×11 k-meshes for the self-consistent and density-of-states calculations,respectively. The Seebeck coefficient was calculated by the Boltzmanntransport theory, assuming constant relaxation time approximationvia the BoltzTraP code.31 The relaxationtime (τ) was determined as the calculated electrical resistivityreproduced the observation.The first-principles band calculation was performed for RbZn3x4–CuxAs3 (0.00 ≤ x ≤ 0.04) samples were synthesized (4S3-type structure (space group P4/mmm). These patterns were analyzed by the Rietveld method . From the results, only a small amountof ZnO was identified as the impurity phase, with a mass fractionof less than 1.5 wt %. Lattice constants a and c were approximatelyindependent of the doping  was estimated tobe Eg = 0.58 eV by fitting the data above595 K using the following equation: ρ = ρ0exp(Eg/2kBT), where kB and ρ0 denotethe Boltzmann constant and a scaling factor, respectively. The semiconductingbehavior disappeared above x = 0.005, followed bythe dramatic suppression of the electrical resistivity with doping.The value of the x = 0.02 sample was still relativelyhigh (ρ = 149 μΩm at T = 312 K),compared with that of the optimally doped As-based 122-Zintl compoundswhose electrical resistivity was less than 100 μΩm.32 The high electrical resistivity originated from relatively low Hallmobilities of 9.65 and 3.84 cm2/(V s) for x = 0.00 and 0.02, respectively, at room temperature [inset of x = 0.02 at T = 300 K on the calculationbased on the Boltzmann transport theory. This indicates that the relativelylow Hall mobilities are due to intense scattering of carriers.The low perature4. This vinset of 4b. The dT = 550 K, after which it decreased, suggesting the intrinsicexcitation of electrons at high temperatures, as implied by the electricalresistivity. The positive values of the Seebeck coefficient indicatedthat the carriers were holes. The values were drastically suppressedby a small amount of Cu doping, becoming 206 μV/K at T = 312 K for x = 0.02. The approximatelylinear increase with heating at x ≥ 0.005was a typical behavior of heavily doped semiconductors.The Seebeck coefficient of the nondoped samplewas high,correspondingto its relatively low carrier concentration 5. It exhS2/ρ) are demonstrated in x = 0.00was rather low, owing to its large electrical resistivity. The powerfactor increased when the electrical resistivity decreased with doping.The value around room temperature was almost the same for the x ≥ 0.01 samples, where it was approximately 0.3mW/mK2. The maximum value of S2/ρ = 0.52 mW/mK2 was obtained in the x = 0.02 sample at T = 797 K.The temperature dependencies of the power factor wascalculated using the Wiedemann–Franz law described as κe = LT/ρ, where L =2.44 × 10–8 WΩ/K2 is the Lorenznumber. The lattice thermal conductivity was determined by subtractingκe from κ. The small value of κe, which was derived from relatively high resistivity, produced almostthe same value of κL with κ. The lattice thermalconductivity independent of doping indicated that the degree of randomnessvia Cu doping was too low to affect the thermal flow.The total thermal conductivity was constant withdoping,exhibitinga significantly low value of 1.67 W/mK at  = 313K 7. The vaT =314 K increased with the doping until x = 0.01, reachingZT = 0.06 for x = 0.015. Temperature dependenciesof ZT demonstrated higher ZT values upon heating because of the higherpower factor and lower total thermal conductivity at higher temperatures,as well as the factor T. The maximum ZT value of0.53 was observed in x = 0.02 at T = 797 K.The doping dependence of the dimensionless figure-of-meritwasmainly determined by the power factor because the total thermal conductivitywas constant with doping. The nondoped sample exhibited a considerablylow ZT value, owing to its high electrical resistivity 8. A sligx = 0.00 sample via theRietveld method . The Zn–As1 bond length is 2.50Å, which is comparable to those of a typical covalent Zn–Asbond (2.48 Å).35 The Zn–As2bond length deviates from the typical value to 2.66 Å. This impliesthe formation of an unusual interatomic potential in the ZnAs4-tetrahedron.The distorted ZnAs4As3 are shown in 38 The calculatedband gap energy was Eg = 0.69 eV, consistentwith that obtained from the electrical resistivity. The band structurecalculated without including the spin–orbit coupling showedthat the first- and second-highest valence bands were degenerate asthey originated from the As px and py orbitals . Moreover, the top of thethird-highest valence band constructed from the As pz orbital was positioned near the Fermi level. However, by consideringthe spin–orbit coupling, the top of the two bands composedof As p orbitals around the Γ point slightly split with a valueof 0.04 eV (corresponding to 4 kBT at 800 K.The calculated band structure and densityof states of RbZn2 atroom temperature was calculated at the carrier concentration of 2.5× 1020 /cm3, which agreed reasonably wellwith the observations (inset of x = 0.02 is close to the optimumvalue.The calculated Seebeckcoefficient as a function of the carrierconcentration at room temperature is demonstrated in the inset of inset of 6. This s4 tetrahedron. The ZnAs4 tetrahedron of the present RbZn4As3 was slightly distorted with three As–Zn–As bond anglesof 113.3, 109.9, and 103.4°. A higher power factor might be expectedif the distortion can be suppressed. Another strategy for obtaininghigher degeneracy is to suppress the spin–orbit coupling. Comparedto As-based compounds, P-based compounds are ideal candidates owingto their sufficiently weak spin–orbit coupling, producing considerablysmall energy splits. The wide variety of 143-Zintl compounds allowsfor the exploration of the most suitable compounds, exhibiting highthermoelectric properties.To improve the power factor, forming higher degeneracyat the topof the valence band is a promising strategy. A high degeneracy ofthe As p orbitals can be obtained in a high-symmetry surrounding thatis achieved by regular ZnAs4x4–CuxAs3 synthesized bysolid-state reactions was investigated. The low lattice thermal conductivitywith a value of 1.67 W/mK at T = 313 K can be causedby the large vibration of the Rb atoms. The maximum power factor andZT were 0.52 mW/mK2 and 0.53 for x = 0.02at T = 797 K, respectively. The value is the highestin 143-Zintl compounds, which opens a new class of high-performancethermoelectric materials.Thethermoelectric performance of RbZn