S. aureus in planktonic and biofilm phase, the activity also depending on the guanosine and cyclodextrin ratio within the hydrogel structure.Facile method for the preparation of β–cyclodextrin–functionalized hydrogels based on guanosine quartet assembly was described. A series of seven hydrogels were prepared by linking β–cyclodextrin molecules with guanosine moieties in different ratios through benzene–1,4–diboronic acid linker in the presence of potassium hydroxide. The potassium ions acted as a reticulation agent by forming guanosine quartets, leading to the formation of self–sustained transparent hydrogels. The ratios of the β–cyclodextrin and guanosine components have a significant effect on the internal structuration of the components and, correspondingly, on the mechanical properties of the final gels, offering a tunablity of the system by varying the components ratio. The insights into the hydrogels’ structuration were achieved by circular dichroism, scanning electron microscopy, atomic force microscopy, and X–ray diffraction. Rheological measurements revealed self–healing and thixotropic properties of all the investigated samples, which, in combination with available cyclodextrin cavities for active components loading, make them remarkable candidates for specific applications in biomedical and pharmaceutical fields. Moreover, all the prepared samples displayed selective antimicrobial properties against  Hydrogels represent complex three–dimensional cross–linked networks that can retain large amounts of water while preserving their structure ,2,3. TheIn our previous works, we have reported the preparation of guanosine–based supramolecular hydrogels with high water content as scaffolds for cell growing applications ,30. WateIn the current work, we describe facile synthesis, characterization, and antimicrobial properties of β–CD functionalized supramolecular hydrogel “reticulated” by the formation guanosine quadruplexes (G4–CD). The presence of seven pairs of 1,2–diols in the structure of β–CD allows for the controlled attachment of up to seven guanosine units by utilizing benzene–1,4–diboronic acid linker, thus offering a tuning tool for the final hydrogel properties studied by rheological measurements. The number of attached guanosine moieties strongly affects the internal structure of the hydrogels observed by the circular dichroism (CD), scanning electron microscopy (SEM), and atomic force microscopy (AFM) studies. Besides, multiple hydrogen bonding throughout the system, tuned by the number of guanosine molecules, offers the thixotropic and injectable properties to the final hydrogel. Additionally, all the prepared hydrogels were assessed for antimicrobial activity against eight reference strains in view of the potential use of the described systems for biomedical applications.Seven G4–CD hydrogels were synthesized in two–step procedure , startinin situ, maintaining the same temperature value. At this step, the 1,2 diols of the guanosine sugar moiety reacted with the free boronic acid group of the linker to yield the designed guanosine–functionalized β–CD units according to The boronic ester formation with the 1,2 diols of the β–CD lower rim occurred within minutes at elevated temperature (90 °C) in the presence of an equivalent amount of the base (KOH) to allow the interaction of only one boronic acid moiety with cyclodextrin diols. Subsequently, to the obtained transparent solution, the equivalent amount of guanosine and KOH was added As previously reported ,32, the To gain insight into the formation of the G4–CD_1–7 hydrogel internal structure, we recorded circular dichroism (CD) spectra of each sample taken directly from the reaction mixture in cuvettes with the lightpass of 0.1 mm. Moreover, the CD spectra of warm samples (60 °C) were recorded every five minutes during cooling down to room temperature and stabilization (55 min) to observe the temperature dependent structural changes in the range between 220 and 340 nm .Although all the investigated samples displayed individual behavior depending on the amount of guanosine moieties attached to the β–CD molecule, similarities were common for all samples . Thus, aIn the 220–290 nm range, negative broad band was observed around 250 nm in all the investigated samples at room temperature . In the The CD bands maxima observed for all the investigated samples are in disagreement with the previously reported data for the telomeric sequences and other G–rich species ,45, exhiThe morphology of G4–CD_1–7 hydrogels was studied by SEM on lyophilized hydrogel samples . The invThe decrease in pore size with the increase in guanosine content of the sample can be observed in all samples except G4–CD_2 . This saTo further investigate the assembly mechanism of the G4–CD_1–7, we performed the structural analysis by X–ray diffraction (XRD). Hydrogels G4–CD_1–7 were lyophilized to form white powders, and X–ray diffraction analysis was performed using CuKα–emission in the angular range 2–50° (2θ) with a scanning step of 0.01° and a recording rate of 2°/min. The data obtained are represented in o and the absence of the characteristic crystalline β–CD diffraction peaks [The XRD data for all the investigated lyophilized samples showed the presence of two main reflections (18.7° and 26.6°) that correspond to the intermolecular d–spacing 3.3 and 4.7 Å, respectively . The preon peaks  indicateon peaks ,49. Typion peaks .The peak at 26.6° represents a characteristic peak describing the π–π distance between G–quartet stacks in previously reported related systems ,32. The The XRD data strongly support the circular dichroism assumptions on the formation of large aggregates based on the G4 structure and aligned β–CD arrangements. To further confirm the formation of long assemblies, we performed detailed AFM investigations of the G4–CD_1–7 samples.Insights on morphological structures of hydrogels G4–CD_1–7 were acquired by AFM. All investigated samples presented inter–connected fibrils of several micrometers in length, with different fibril thickness .Since this type of structuring was not observed on previously reported G4 hydrogel systems , we assuAll the investigated G4–CD_1–7 samples revealed gel–like behavior, characterized by storage modulus (G’) higher than loss modulus (G”) a.c), located in the range of 150–250%, when their structure was broken, and they acquired liquid–like properties with G” > G’. The structures started to change after a limit strain (γl), which did not significantly depend on the β–CD content  values, corresponding to γl and γc, were around 2 Pa (τl) and 16 Pa (τc), respectively. The interactions between the components of the investigated systems (which gave the gel strength) were weak, and at a rather low value of τl, the network structure started to break.The samples were stable up to a critical strain (γ content b. The γlThe differences between the viscoelastic moduli of the investigated samples were not significant . Thereby2 to 10–1 Pa·s) by increasing –2 to 102 s–1. The values of viscosity at low shear rate (η0) were determined by fitting the experimental data  with the increase of shear rate, au model  . In order to investigate the structural recovery degree, the samples were subjected to a rheological test with three shear steps: (1) the samples were submitted for 5 min to a strain of 1% (from LVR); (2) a strain of 300% (outside LVR) was applied for the next 5 min; (3) the structure recovery was followed, to the strain of 1% for 12 min.At the end of the test, the structures of samples with high β–CD content were fully recovered, proving that β–CD presence favored the restoration of the interactions between the system components. In addition, these samples showed a slight increase of viscoelastic moduli under reduced shear, probably because of the new interactions formed d. After For the G4–CD_7 hydrogel, an experiment to visualize the injectable properties was performed; the results are provided in the video file (V1) in the Since the designed G4–CD systems were envisioned for biomedical applications, it was important to assess their antimicrobial properties. The antimicrobial ability of G4–CD_1–7 was evaluated against eight reference strains; the results are summarized in S. aureus.All the hydrogels did not present antimicrobial activity against most of the investigated reference strains but were efficient against the Gram–positive strain represented by S. aureus could be explained by the specific action of substituted CDs over the integrity of Gram–positive bacteria membranes observed earlier [+ efflux through definite organizations in bacterial membrane, leading to subsequent complete loss of the K+ [S. aureus in planktonic and biofilm phase , the higher the observed destroying potential of the hydrogel.According to The described hydrogel systems can potentially be loaded with dedicated antibiotics able to form stable inclusion complexes with cyclodextrins ,55,56. TThe chemicals utilized in this work such as β–CD, benzene–1,4–diboronic acid, guanosine, fluoresceine, LiOH and KOH were purchased from Sigma . All the purchased reagents were used without further purification. Ultrapure water was used throughout the experiments and stock solutions preparation.Synthesis of G4–CD hydrogels was performed by adapting the previously reported synthesis of guanosine–based hydrogels ,30. DepeFor hydrogels G4–CD_2 to G4–CD_7, the total volume of the reaction mixture was maintained at 2 mL and the quantities of all components were constant except for the β–CD. The necessary equivalents of β–CD for the corresponding ratios were adjusted by the volume of β–CD stock solution in the reaction mixture .The morphology of G4–CD_1–7 hydrogels was studied by SEM and AFM techniques. In the case of SEM , hydrogel samples were freeze dried prior to measurements, carefully placed onto 25 aluminum plates with double–sided adhesive and investigated at an accelerating voltage of 20 kV.−1, and a tip curvature radius of 10 nm were used. Aliquots of 10 µL gel solution were evenly deposited onto freshly cleaved mica substrates and dried in air at room temperature prior to imaging. AFM image analysis was performed with Gwyddion 2.59 software [AFM investigation was conducted using Ntegra Spectra Atomic Force Microscope  operated in tapping mode under ambient conditions. Silicon cantilever tips  with gold reflecting coating, a resonance frequency of 140–390 kHz, a force constant of 3.1–37.6 N msoftware .−1 acquisition speed in 1 mm lamellas. Typically, 100 µL of sample from the hydrogel preparation reaction mixture (90 °C) was poured directly onto a lamella and covered with the second glass to form a uniform film with no air bubbles in between. All samples were recorded directly from the reaction mixture at the starting temperature of 60 °C, with measurements performed every 5 min until stabilization (55 min).Circular dichroism (CD) measurements were conducted on a Chirascan plus . The CD spectra were recorded between 340 and 220 nm with data pitch 0.5 nm at 200 nm·minHydrogels G4–CD_1–7 were lyophilized to form white powders and X–ray diffraction analysis was performed on a Rigaku Miniflex 600 diffractometer  using CuKα–emission in the angular range 2–50° (2θ) with a scanning step of 0.01° and a recording rate of 2°/min.−1, in the strain (γ) range of 0.05–500% (the shear stress between 0.01–30 Pa). The storage modulus (G’), the loss modulus (G”), and the loss tangent (tan δ), defined as G”/G’ at 1 Pa, were established. The flow curves were determined from measurements in continuous shear regime in the range of shear rates . The rheometer is equipped with a Peltier system, which ensures the temperature control, and a solver trap cover to prevent the solvent evaporation that is used. In order to establish the linear viscoelastic regime (LVR), an amplitude sweep test was realized at a constant oscillation frequency (ω) of 10 rad s® AQueous One Solution Cell Proliferation Assay from Promega, were purchased.For the microbiological assay: Tryptic Soy Broth, Nutrient Broth, and bacteriological agar from VWR Chemicals, Sabouraud Dextrose Broth from Merck, and CellTiter 96Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Enterococcus faecalis ATCC29212, Klebsiella pneumoniae ATCC10031, and Salmonella typhimurium ATCC14028, yeast strains represented by Candida albicans ATCC10231 and Candida glabrata ATCC90028, and the fungal strain Aspergillus brasiliensis ATCC9642.The antimicrobial activity of the hydrogels was determined by disk diffusion assay  against All microorganisms were stored at −80 °C in 20–40% glycerol. The bacterial strains were refreshed in tryptic soy broth (TSB) at 37 °C. The yeast strains were refreshed Sabouraud dextrose broth (SDB), and the fungal strain was refreshed on potato dextrose broth (PDB) at 25 °C. Microbial suspensions were prepared with these cultures in sterile solution to obtain turbidity optically comparable to that of 0.5 McFarland standards. Volumes of 0.2 mL from each inoculum were spread on the Petri dishes. The hydrogels were placed on proper agar plates and 100 µL of the samples was added in 5 mm holes cut into agar media.To evaluate the antimicrobial properties, the growth inhibition was measured under standard conditions after 24 h of incubation at 37 °C for the bacterial and the yeast strains and after 48 h at 26 °C for the fungal strain. All tests were conducted in triplicate to verify the results. After incubation, the diameters of inhibition zones were measured by using Image J version 1.52 t software .S. aureus, in planktonic (free floating) phase and after biofilm formation, was assessed by MTS assay using the CellTiter 96® AQueous One Solution Cell Proliferation  according to the manufacturer instructions.The hydrogels bacterial cytotoxicity after incubation with the ® Omega microplate reader .Briefly, after 24 h of bacterial incubation with the tested hydrogels, MTS reagent was added 1–3 h prior to absorbance readings. After the formazan formation, the final reading was performed at 490 nm on a FLUOstarAll data were expressed as the mean ± standard deviation of the mean. Statistical analysis was performed using XLSTAT Ecology version 2019.4.1 software .S. aureus in planktonic and biofilm phase, the activity also depending on the guanosine and cyclodextrin ratio within the hydrogel structure.We reported the design and preparation of seven cyclodextrin containing supramolecular hydrogels whose gelation mechanism is based on the formation of guanosine quadruplexes, thus maintaining the cyclodextrin availability for the possible host–guest interactions. The covalent insertion of cyclodextrin into the hydrogel matrix was performed by the reaction of 1,2 diols of the β–CD lower rim with the benzene–1,4–diboronic acid linker and subsequent attachment of guanosine molecule to the linker. The presence of potassium ions in the reaction mixture led to the arrangement of the guanosine moieties into G4 structures and, correspondingly, to the formation of transparent hydrogels. The obtained hydrogels’ properties strongly depend on the number of guanosine units attached to the cyclodextrin molecules, thus offering a property tuning tool for the possible applications. The increase in the amount of guanosines led to the increase in internal structuration observed by AFM, SEM, and XRD analyses. Additionally, the ratio between guanosine and cyclodextrin in the hydrogel structure strongly influenced the rheological properties of the sample, while the supramolecular nature of the samples offered interesting self–healing and thixotropic properties. All the prepared samples displayed selective antimicrobial properties against


Here, it is reported that the ferromagnetism in two and five unit cells thick LMO films epitaxially deposited on (001)‐SrTiO3 substrates, a ferromagnetic/ferromagnetic coupling in eight and ten unit cells ones, and a striking ferromagnetic/antiferromagnetic pinning effect with apparent positive exchange bias in 15 and 20 unit cells ones are observed. This novel phenomenon in both 15 and 20 unit cells films indicates a coexistence of three magnetic orderings in a single LMO film. The high‐resolution scanning transmission electron microscopy suggests a 1/nP2 to Pbnm symmetry transition from interface to surface, with the spatial stratification of MnO6 octahedral morphology, corresponding to different magnetic orderings. These results can shed some new lights on manipulating the functionality of oxides by interface engineering.A variety of emergent phenomena are enabled by interface engineering in the complex oxides heterostructures. While extensive attention is attracted to LaMnO 1/nP2 structure has only been predicted theoretically in LaMnO3 films previously. This work demonstrates the experimental observation of the emergent magnetic orders including ferromagnetism and antiferromagnetism, strongly correlated with the octahedral distortion in 1/nP2 and Pbnm structures in different layers of LaMnO3 ultrathin films, respectively, which opens a window for engineering functionality via interfacial symmetry breaking.Unconventional  Stoichiometric LMO bulk is known to be a layer‐type (A‐type) antiferromagnet, in which Mn3+ is a Jahn–Teller ion with a t2g3eg1 occupancy and the in‐plane interaction between adjacent Mn ions is ferromagnetic (FM) while the out‐of‐plane one is antiferromagnetic (AFM). In contrast to bulk LMO, there has been lots of controversy about magnetic phase in thin films. While some studies describe the appearance of FM behavior in stoichiometric thin films, other reports have shown that AFM order staying in the films thinner than 5 unit cells (u.c.), and FM phase in films thicker than 5 u.c. Meanwhile, almost all experiments have unveiled an insulating feature for LMO films in both FM and AFM phases, although many theoretical reports have predicted that LMO thin films possess a Pbnm structure and with a ferromagnetic metallic phase. Numerous efforts have been devoted to eliminate this paradox by reducing sample symmetry or confirming electronic phase separation, among which Xiang et al. first introduced unconventional 1/nP2 structure in LMO to explain this ferromagnetic insulating feature. However, this novel 1/nP2 structure in LMO films has not been experimentally observed so far.Artificial oxide heterostructures with chemically abrupt interfaces provide a platform for engineering bonding geometries that lead to emergent phenomena.7, 8ferromagnetic insulating state in ultra‐thin LMO films with thickness of 2 and 5 u.c. We demonstrate a FM/FM coupling in 8 and 10 u.c. films, indicating the appearance of a harder FM state in the region above 5 u.c. In addition to the FM/FM coupling, a striking FM/AFM pinning effect with remarkable positive exchange bias is shown in 15 and 20 u.c. thin films, indicating an AFM order appearing in the layer above 10 u.c. away from the interface. To reveal the evolution of magnetic property in different regions, we show the characterizations by high‐resolution scanning transmission electron microscopy (STEM), which displays a transition from 1/nP2 to Pbnm structure, corresponding to the evolution of FM to AFM. Our study demonstrates that different magnetic orderings, diverse octahedral morphologies, and various exchange couplings can be achieved in a single oxide by controlling their dimensionality.In this paper, we first report an experimental observation of 2Figure ab plane. Besides, all samples present lattice relaxation along the c direction  patterns in n Figure –f.Figure  This FM feature has also been found in 5 u.c. samples displayed in Figure Ms). This superimposition indicates that two different FM layers present in these films. For the simplicity to be mentioned later, we name it as the first ferromagnetic layer (FML1) with thickness roughly goes from 1 to 6 u.c. The second ferromagnetic layer (FML2) means the layer with harder FM feature (with larger coercive filed) approximately from the 7 to 11 u.c. Besides, the FM/FM interaction between FML1 and FML2 brings a distinct exchange spring effect in these films. This step‐like feature retains in 15 . The existence of this AFM order is also verified by the abrupt drops in the MH loops around zero magnetic field, indicated by red circles  in 15 (200) u.c. films from that in 10 (15 and 20) u.c. films shown in Figure 5 Figure , 20 , Mn (L‐edge), and O (K‐edge), respectively, of LMO films with different thicknesses. A direct comparison from the electron energy loss spectroscopy (EELS) spectra collected for La, Mn, and O, respectively, of a 15 u.c. LMO film is shown in Figure L3 edge of Mn from interface to surface  and LMO. It is known that the net charge imbalance between two adjacent sublayers will inevitably lead to the polar catastrophe. Therefore, the charge transfer between two transition metal ions happens to neutralize the charged interfaces. For an ultrathin LMO film with a thickness below 2 u.c., we believe that surface effect may also contribute to add free electrons, e.g., oxygen vacancies or strain‐induced nonstoichiometry, into LMO film, leading the partial Mn3+ ions change to Mn2+ ions.Figure Figure  where the octahedra manifest vast diversity from each layer. We carried out statistics over 50 atomic columns and 22 atomic rows from the STEM image shown in Figure 6 octahedra and belongs to typical Pm3¯m space group. In both FML1 and FML2, two kinds of octahedra, denoted as OC1 and OC2, appear alternatively in G type manner with 3D alternation of OC1 and OC2. Figure LO—O) in ab plane (xL) and c axis (zL) of the octahedra denoted as OC2 for one row and OC1 for the nearest rows. zL (xL) is always longer (shorter) in OC1 than the one in OC2, shown as the zigzag curve in Figure c‐axially elongated and zL is longer than xL for FML1 due to the compressing effect from the substrate. This characteristic is verified by the statistics of STEM characterization for 5 u.c. LMO films displayed in Figure zL is longer (shorter) than xL in OC1 (OC2). The alternation of OC1 and OC2 in FML1 matches perfectly with the octahedral spatial arrangement of LMO in the 1/nP2 structure predicted by Xiang et al. Further evidence on the octahedra tilt is illustrated by systematic analysis of Mn—O—Mn bond angle (θMn—O—Mn) across the film growth direction  and LMO (orthorhombic). The relaxation of shear strain will inevitably change the crystallographic symmetry of LMO films with increasing thickness. This substrate/film lattice and symmetry mismatch is the main source of the octahedral rotation modification and Jahn–Teller distortion. The evolution of MnO6 octahedra changes bond length and angle, thus affecting the balance between the intra‐atomic exchange interaction energy and crystal field energy through the structural distortion. As a result, the electronic ground state is modified, leading to the observed emerging magnetic phenomenon in our LMO films.To reveal the mechanism of our magnetic results, the unit‐cell‐resolved structural evolution is characterized with high‐resolution STEM. Figure 31 whexL (zL) remains roughly the same for OC1 and OC2, with zL being longer than xL. The similarity of OC1 and OC2 in AFML with c‐axially stretching and ab plane compressing remains, which is described as normal Q3 distortion mode. The octahedra in AFML manifest the heaviest rotation ‐oriented STO substrates at 680 ℃ using a pulsed laser deposition (PLD) system. During the deposition, the target‐substrate distance was set to 7.2 cm and the oxygen partial pressure was adjusted to 0.1 Pa. A XeCl laser with central wavelength of 308 nm was employed to provide a laser energy density of 1.2 J cmThe XRD and RSM analyses were performed using a Rigaku SmartLab (8 kW) high‐resolution X‐ray diffractometer, with the wavelength of the X‐ray 0.154 nm. The XAS was collected under the total electron yield (TEY) mode at the 4B9B line station of Beijing Synchrotron Radiation Facility.ab plane when measuring MH loops at 10 K. The resistivity was measured using van der Pauw methods with PPMS.The magnetic properties of these samples were measured with a vibrating sample magnetometer (VSM) of physical properties measurement system (PPMS). The external magnetic field applied was from −3 to 3 T parallel to Cs) correctors.The atomic structures of these heterostructures were characterized using an ARM‐200CF transmission electron microscope operated at 200 keV and equipped with double spherical aberration  in each octahedron and the M—O—M angle (θM—O—M) between adjacent octahedra were calculated. As the LMO sample showed alternation of OC1 and OC2, the O—O length and θM—O—M were collected every other column. The dots in Figure a axis. The four left (right) bar charts represented the O—O length on odd (even) atomic columns.The STEM images are imported in Figure The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The recognition of Archean microbial methane in this work reveals a biochemical process of greenhouse gas generation before the Great Oxidation Event and improves the understanding of the carbon and hydrogen geochemical cycles.Methane, along with other short-chain alkanes from some Archean metasedimentary rocks, has unique isotopic signatures that possibly reflect the generation of atmospheric greenhouse gas on early Earth. We find that alkane gases from the Kidd Creek mines in the Canadian Shield are microbial products in a Neoarchean ecosystem. The widely varied hydrogen and relatively uniform carbon isotopic compositions in the alkanes infer that the alkanes result from the biodegradation of sediment organic matter with serpentinization-derived hydrogen gas. This proposed process is supported by published geochemical data on the Kidd Creek gas, including the distribution of alkane abundances, stable isotope variations in alkanes, and CH 4 kept the early Earth warm under the faint young sun, but clear records are lacking. Here the authors present isotopic evidence that CH4 seepage in the Canadian shield is from hydrogen biodegradation in a Neoarchean ecosystem rather than an abiotic synthesis product.Microbial CH The generation of microbial methane, as a significant step of the global carbon cycle, is reflected in the stable isotopic compositions of organic matter. However, finding and interpreting records that span billions of years are challenging3. Over the past two decades, methane and other short-chain alkane gases discovered in ancient shields have shown broad variations in hydrogen isotopic compositions (δD), but relative uniformity in carbon isotopic compositions (δ13C)5. The first reported example is from the Kidd Creek mines in the Neoarchaean Abitibi Greenstone Belt of the Canadian Shield near Timmins, Ontario, Canada4. In this area, multiple cycles of sedimentation, volcanic activities, magma intrusions, and serpentinization of mafic rocks occurred ~2.7 Ga6. A Neoarchaean ecosystem has been indicated by biomarker evidence7. The Kidd Creek mines produce metal sulfides associated with underground felsic volcanic rocks8; alkane gases (from methane to pentanes) have been discovered at depths of 2000–3000 m in exploration boreholes4. Reported gas contents and isotopic compositions show, in addition to the above δD and δ13C patterns, an exponential distribution of alkane abundance and deuterium–deuterium clumped isotopic composition of methane (Δ12CH2D2) between −10 and 5‰ (for samples collected <400 days since the borehole was drilled)11.Microbial methane was an important source of greenhouse gases that kept the early Earth warm before the Great Oxidation Event  under the faint Sun13C patterns are different from those found in alkanes generated from the decomposition of organic matter4. However, these patterns have neither been found in hydrothermal gas, commonly believed as abiotic12, nor in artificial FT synthetic gas13. Therefore, the abiotic interpretation is empirical and lacks a chemical rationale, especially when the feasibility of this synthesis under geological conditions has been questioned in recent years16. Moreover, the abiotic interpretation overlooked the possibility of forming the unique isotopic distributions in a distinct oxygen-depleting Archaean ecosystem.Upon its discovery, the Kidd Creek gas was interpreted as abiotic through Fischer–Tropsch (FT) synthesis because the δD and δ2, while the rest are inherited from long alkyl chains. The distributions of alkane abundances, carbon, hydrogen, and clumped isotopic compositions suggest that the gas was generated through hydrogen biodegradation of sediment organic matter. The Kidd Creek gas is a record of microbial greenhouse gas generation before the GOE.In this work, we present the evidence that the Kidd Creek gas is a microbial product in a Neoarchaean ecosystem. From the deuterium distribution in short alkanes, we discover that the hydrogen atoms in each short alkane molecule come from two sources: two capping H atoms are donated by serpentinization-derived H13C and δD of alkanes between the Kidd Creek gas, typical thermogenic gases18, and gases from a hydrothermal field12. The unique contrastive alkane δ13C and δD variations in the Kidd Creek gas indicate that the hydrogen isotopic distribution is unlikely due to a pure kinetic isotope effect (KIE) during its formation. Instead, the wide δD variation implies that the hydrogen atoms likely have multiple sources with distinct δD values. The variation suggests a reaction involving the conversion of organic matter to short-chain alkanes in the presence of H2, a hydrogen degradation (or hydrogenolysis) reaction. The exponential distribution of alkane gas abundance with respect to carbon number , the amount of methane generated from branched primary carbons is equivalent to the amount generated from tertiary carbons; the average number of H atoms from H2 is still 2. As a result, the hydrogen isotopic composition of the generated alkane CnHn+22 is expressed asDuring hydrogenolysis, long alkyl chains (straight or branched) are consumed segment by segment consecutively. Each generated short-chain alkane molecule may contain 1, 2, or 3 capping hydrogen atoms from Hnds Fig. . Most aln + 2). Similar analysis has successfully explained the linear variation between δ13C and 1/n in thermogenic alkanes20.In Eq. , subscriR2 = 0.98) indeed exists in the Kidd Creek gas  = −1130‰ (hence δDB = −691‰). The δDA and δDB values are close to the δD values of marine organic matter (−80 to −170‰)21 and serpentinization-derived H2 in the Kidd Creek gas (~−730‰)22, respectively. Therefore, the reaction between the long alkyl chains in organic matter and serpentinization-derived H2 explains the δD isotopic pattern of the Neoarchaean alkane gas. The slight difference of δDB > δDH2 indicates a weak and inverse deuterium KIE (DKIE) during some reaction steps . The linear trends with high coefficients of determination in the Kidd Creek gas  with H2 also suggests a catalytic process. The reason is that a C–C bond (bond energy 346 kJ/mol) is more vulnerable to crack than an H–H bond (bond energy 432 kJ/mol). If there were no catalytic processes to lower the activation barrier of H–H splitting, C2+ gases would be more depleted. Because abiotic catalysis requires dispersed elemental transitional metal catalysts without thermal-sintering and sulfur-poisoning25, a demanding condition hardly satisfied in geological bodies, the catalytic process revealed here is most likely a microbial process.In contrast to the hydrogen atoms, which have two sources, all the carbon atoms in the Kidd Creek hydrocarbon gases originate from organic precursors. As a result, kinetic isotopic fractionation (instead of multiple sources) is the main reason for the δgas Fig. ; therefo13C KIEs. A CH4 molecule has only one carbon atom, which experienced a primary 13C KIE during C–C cleavage. Other alkane molecules have primary carbon atoms and carbon atoms adjacent to the primary ones. The primary carbon atoms experienced a primary 13C KIE during the cleavage; their adjacent ones experienced a secondary 13C KIE. The combination of a normal secondary 13C KIE and an inverse primary one may form the above δ13C order, as demonstrated by a numerical simulation on the consecutive random scission of long alkyl chains . However, hydrogen atom exchange between methane molecules requires an elevated temperature and the existence of catalysts ; under these conditions, ethane and propane would start to decompose 29. The high fraction of C2+ alkanes (~15 vol%) in the Kidd Creek gas rules out a significant hydrogen exchange between methane isotopologues. A more concrete explanation for the above contrast is that clumped isotopic distribution during methane generation is normally kinetically governed, and an excessive KIE due to D–D clumping may form the observed Δ12CH2D2 value30. To demonstrate this explanation, we conducted kinetic numerical simulations on bulk and clumped isotopic fractionations of CH4 generation , which provides further support that the gas was formed through an enzyme-catalysed microbial process26.Because δD variation in alkanes is mostly determined by the contributions of the two hydrogen sources with distinct δD values, the DKIE during this microbial reaction is hardly evaluable based on δD values. However, the DKIE can be evaluated from the deuterium–deuterium clumped isotopic composition Δ2) using alkyl chains suggests an abnormally low oxygen fugacity in the ecosystem, where electron acceptors such as O2, Fe3+, SO42−, and even CO2, which are more efficient than alkanes to oxidise H2, are absent. This anoxic ecosystem could have been widely present either before the GOE or only present in the deep earth after the GOE when the Earth’s surface was too oxidative. By comparing hydrocarbon gases from various serpentinization sites  were obtained from the sites with both pre-GOE serpentinization and pre-GOE organic matter sedimentation  brought about the graphitisation of organic precursors and the termination of biodegradation conditions32. Other sites include (1) the Kloof, Driefontein, and Mponeng mines in the Witwatersrand Basin of the Kaapvaal Shield (southern Africa), where serpentinization and organic matter sedimentation occurred 3.0–2.7 Ga33, and (2) the Copper Cliff mines in the Sudbury Basin of the Canadian Shield, with sedimentary organic matter in the Paleoproterozoic Huronian Supergroup deposited ~2.45 Ga34, just before the onset of the GOE35.All previously reported hydrocarbon gas samples with the above alkyl hydrogenolysis features  to the present , the isotope pattern of hydrogenolysis in the gas eventually becomes altered. The δD and Δles Fig. .Hydrogenotrophy , methanotrophy, and the oxidation of higher alkanes. The oxygen-deficient Archaean ecosystem was crucial for the hydrogen biodegradation and the unusual isotopic signatures. Before the GOE, the atmospheric O2 partial pressure was <0.001% or even 0.00001% of the present atmospheric level (PAL); sulfate concentration in the ocean was 2.4% of the current level38. The oxygen-deficient atmosphere and ocean prevented both biological and chemical oxidation of organic matter in sediments39. Therefore, the Kidd Creek gas is a chemical fossil of the pre-GOE ecosystem preserved in the tectonically stable Canadian Shield.The above observations show that the occurrence and preservation of isotopic signatures from the hydrogenolysis reaction require a narrow chemical condition and specified microbial species, which are hardly satisfied after the GOE when strong oxidisers and diversified microbes coexist from shallow to deep earth. These signatures are vulnerable to post-GOE microbial reactions, such as methanogenesis from CO13C depletion in solid organic matter in some Archaean metasedimentary rocks. There are two distinct types of δ13C values in Archaean solid organic matter: one is within the normal range (−26 to −33‰), and the other is more depleted 40. The latter was attributed to methane cycling3, where methane was considered a precursor of the kerogen. Our discovery provides a more plausible explanation: kerogen (and its precursor) in some Archean sediments may have undergone microbial hydrogenolysis in the presence of H2. Through this alkyl removal process with a weak and possibly inverse 13C KIE, the residual kerogen  may have been depleted of 13C and side alkyl chains. Due to the lack of side alkyl chains, the residual kerogen would not have experienced 13C enrichment by thermal dealkylation; it would have kept the 13C-depleted signature.The microbial hydrogenolysis process explains the τ) for diffusion is inversely proportional to diffusivity41. Diffusive fractionation starts to have a significant impact on the isotopic compositions of residual gas when the preservation time is close to the characteristic time42. The sealing rocks of the Kidd Creek gas are crystalline rocks with gas diffusivity (<10−15 m2/s) three orders of magnitude lower than tight shale (10−12 m2/s)44, meaning that τ of the Kidd Creek gas-bearing rocks is 1000 times longer than that of the typical Palaeozoic gas reservoir in shale rocks, compensating their 10-fold difference in the preservation time. There is no significant impact from diffusive fractionation through shales on the isotopic compositions of the Palaeozoic natural gas accumulations44; therefore, the residual Kidd Creek gas unlikely experienced remarkable diffusive fractionation.Comparison between the Kidd Creek rocks and typical Palaeozoic natural gas reservoirs on their diffusion time scales. The characteristic time scale  raises the question of whether the isotopic compositions of the residual gas were affected by diffusive fractionation. This impact is insignificant according to the two comparisons:2. Considering bond energies of 346, 411, and 432 kJ/mol for C–C, C–H, and H–H bonds, hydrogenolysis of long-chain alkanes is an exothermic reaction with a net heat release of 44 kJ/(mol H2). In contrast to the modern hydrogenotrophic methanogenesis of oxygen-bearing compounds45, microbial hydrogenolysis of long alkyl chains is a different geochemical pathway to convert H2 and acted as a significant source of greenhouse gas before the GOE, when the young Earth experienced widespread serpentinization of mafic volcanic rocks in the high heat-flow lithosphere. Conversion of organic matter and the serpentinization-derived H2, along with the hydrogen isotopic distribution in the ecosystem, are summarised in Fig. Hydrogenolysis of organic matter could act as a pathway for Archaean microbial life to obtain chemical energy stored in Hm (between the no. m and no. m + 1 carbon atoms) of an n-alkyl chain with n carbon atoms (1 ≤ m < n), the hydrogenolysis reaction equation isConsidering the cleavage at position n-alkane molecule isSimilarly, the reaction equation for cleavage on an mHm+12, H-CmHm+12, and H-Cn – mHn-m)+12 are still subject to hydrogenolysis until all C–C bonds break down with CH4 as the ultimate product. The cleavage rate (r) of a kerogen side chain (RCm) or an alkane molecule (HCm) is proportional to the C–C chain length and concentration c:Branched alkyl chains are omitted for simplicity. The cleavage reactions are consecutive; the products R-Ck is the reaction constant to break any C–C bond. For simplicity, the difference in k between a middle and an end bond in the C–C chains is not considered. The net reaction rate of a kerogen side chain with a length of m carbon atoms isHere, t is time and N is the maximum chain length of the reaction system. The first term of the right-hand side accounts for the cleavage of the kerogen side chain, and the second term accounts for the generation of residual shorter side chains from the cleavage of the longer side chain. The net reaction rate of a normal alkane with m carbon atoms isHere, m is generated from cuttings at the m and the n − m positions of HCn.The first term of the right-hand side accounts for the cleavage of this alkane, the second term accounts for the generation of the alkane from the kerogen side chain, and the third term accounts for the generation from the cracking of alkanes longer than this alkane. The factor in the last term is necessary because HCEquations  and 7)  7) show 13C KIEs on the rate constant k are considered. Suppose that there is a 13C substitution at position j (1 ≤  j ≤ n); if j = m − 1 or j = m, then there is a primary 13C KIE, or, if j = m − 2 or j = m + 1, there is a secondary 13C KIE on the cleavage at the m position.Both primary and secondary nmin, a maximum chain length of nmax, a mean length of (nmin + nmax)/2, and a standard deviation of σ. When the mean length is large enough (>15), the isotopic compositions of gas products are insensitive to the initial kerogen side chain length distribution. For initial values, a δ13C value of −35‰ is applied. The initial chain length is in a normal distribution with a peak of C17 and a standard deviation of σ = 2 carbon atoms. The initial alkane concentrations are assumed to be 0.A normal distribution is used for the molar distribution of the lengths of initial kerogen side chains, with a minimum chain length of 13C substitution at any position of any side chain is identical and determined by the initial carbon isotopic composition δ13C. Multiple 13C substitutions on a C–C chain are omitted because consideration of multiple substitutions would drastically increase the modelling complexity. This approximation is valid when the C–C chain is not too long. For example, the ratio between the probabilities of double and single 13C substitution in a C20 chain is 13C/12C ~ 0.01. Such a chain is long enough that the δ13C of gas products is insensitive to C–C chain length. Numerical simulation was conducted with Mathworks MATLAB 2020a.For simplicity, we assume that since there is no isotopic fractionation within or between the alkyl chains at the beginning of hydrogenolysis, the probability of Conversion of methylene in a long C–C chain to methane is generalised into two steps:a) is the conversion of the methylene group R-CH2-R′ to a methyl group (RCH3) by accepting a capping hydrogen atom from the hydrogen donor (activated H2); the second step (step b) is the conversion of the methyl group to methane by accepting another capping hydrogen atom. This scheme is highly generalised, and each step may involve multiple elementary biochemical reaction steps, such as the binding of H2 and long alkyl chains to the enzyme, activation of H–H and C–C bonds, and release of the short alkane products from the enzyme. It is beyond the scope of this work to discuss the detailed biochemical reaction steps. But the cleavage and formation of chemical bonds in these steps should be constrained by the observed isotopic patterns.The first step  in the simulation involving clumped isotopic fractionation, as explained in the following. A conventional kinetic model of the decomposition of organic matter without considering the constraints of C–C chain lengths is a zero-dimensional problem. Modelling the random cutting of long C–C chains without considering isotopes is a one-dimensional problem, and modelling bulk carbon isotopic fractionation during random cutting (Model A) is a two-dimensional problem. If 13CH2D2 or 12CHD3 are ignored due to their low abundances). For the subscripts in Fig. m, i, and j in ramij or rbmij), the first digit (m = 0 or 1) is the number of 13C atoms involved in the reaction, the second digit  is the number of deuterium atoms connected in the methylene or methyl group, and the third digit (j = 0 or 1) is the number of deuterium atoms in the hydrogen donor.Reaction equation Eq. Clumped isotopic compositions of methylene and methane are defined as the following:Note that the isotopic compositions here are expressed in decimals; they should be multiplied by 1000 to give per mil values.The deuterium isotope ratio between the hydrogen donor (denoted with subscript B) and the methylene group (subscript A) is expressed as:kamij for step a or kbmij for step b. Kinetic fractionation factors αamijk = kamij/ka000 and αkbmij = kbmij/kb000 define KIEs. Note that a DKIE is often expressed as kH/kD, which is the reciprocal of the αk nomenclature here. A DKIE may be primary or secondary; a primary DKIE results in αa001k ≠ 1 and αb001k ≠ 1, and a secondary one results in αa010k ≠ 1 and αb010k ≠ 1. Kinetic clumped isotope fractionation factors γamij = αamijk/(αa100kmαa010kiαa001kj) and γbmij = αbmijk/ (αb100kmαb010kiαb001kj) define the excessive KIE due to isotope clumping in steps a and b, respectively30.For each reaction step in Fig. 2-R′ is defined as 1 − f, where f is the residual fraction of R-CH2-R′:Conversion of the reactant R-CHf = 1) is derived as:Considering the isotope abundance of D << H, the analytical solution of isotopic compositions at the beginning of the reaction (f = 0) isIf the abundance of the hydrogen donor is excessive and thus approximately constant, then the analytical solution at the end of the reaction  and the clumped isotopic compositions in the precursor are 0, then clumped isotopic compositions become the following:If the KIE is absent 11, indicating that 13C–D clumping in the methylene precursor should be considered to explain Δ13CH3D, and DKIE should be considered to explain Δ12CH2D2.With 13CCH4, δDCH4, Δ13CH3D, and Δ12CH2D2 values at higher conversions of organic precursors are within the reported ranges.The δA and δDB values are close to the derived values from Fig. CH4 is mainly determined by δD of the precursors rather than by DKIE during the hydrogenolysis.The δDNumerical simulations are carried out to find parameters satisfying the following:13CHDR′ =6‰ in the organic precursor is applied so that the final Δ13CH3D is in the range of reported values. This 13C–D clumping in the precursor is acceptable, considering that Δ13CH3D = 5.6‰ has been reported for biogenic gas11. A Δ12CH2D2 value close to the observed value but much higher than the stochastic one  or an inverse secondary DKIE  is necessary, and through numerical simulation, we found that only the inverse 1° DKIE satisfies the above-mentioned δDA, δDB, and Δ12CH2D2 values.A value of ΔR one Eq.  requires13C fractionation between the organic precursor and the methane product is obtained with the KIE parameters . The δD and Δ12CH2D2 values are independent of 13C KIE. Both the bulk and clumped isotopic compositions of methane within the range of reported values are obtained at the organic precursor conversion of 0.65–0.70 as constrained by Fig. Two scenarios  are modelled Fig. . The parers Fig. . With suent Fig. . Note thPeer Review File


Because of the high kinetic barriers, the reaction usually needs to proceed at higher temperature (>300 °C). High-efficiency CO2 methanation at low temperature (<200 °C) is an interesting topic, and only several noble metal catalysts were reported to achieve this goal. Currently, design of cheap metal catalysts that can effectively accelerate this reaction at low temperature is still a challenge. In this work, we found that the amorphous Co–Zr0.1–B–O catalyst could catalyze the reaction at above 140 °C. The activity of the catalyst at 180 °C reached 10.7 mmol2CO gcat−1 h−1, which is comparable to or even higher than that of some noble metal catalysts under similar conditions. The Zr promoter in this work had the highest promoting factor to date among the catalysts for CO2 methanation. As far as we know, this is the first report of an amorphous transition metal catalyst that could effectively accelerate CO2 methanation. The outstanding performance of the catalyst could be ascribed to two aspects. The amorphous nature of the catalyst offered abundant surface defects and intrinsic active sites. On the other hand, the Zr promoter could enlarge the surface area of the catalyst, enrich the Co atoms on the catalyst surface, and tune the valence state of the atoms at the catalyst surface. The reaction mechanism was proposed based on the control experiments.CO It is discovered that an amorphous transition metal catalyst Co–Zr0.1–B–O could effectively accelerate CO2 methanation, at a rate that is comparable to or even higher than that of some noble metal catalysts under similar conditions. Chemical transformation of CO2 into fuels or useful chemicals has attracted great attention all over the world.1–10 Methane is one of the major energy sources in human life that can be easily fed into the existing infrastructures. In addition, methane is also a basic feedstock to produce other value added chemicals.11–13 Hydrogenation of CO2 into methane, i.e., CO2 methanation, is among the most important topics of CO2 valorization.14,15 CO2 methanation is a reversible and strong exothermic reaction, and is thermodynamically favorable. However, it is difficult to achieve because of the high kinetic barriers of the eight-electron reduction process. Many transition metals such as Ni, Fe, Co, Ru, Rh, and Pd have been investigated as catalysts to accelerate this reaction.16–19 To obtain satisfactory catalytic results, the reaction usually needed to proceed at higher temperature (>300 °C), where the undesired endothermic reverse water gas shift (RWGS) reaction tended to occur. For example, nickel based catalysts with various supports were the extensively studied catalysts for CO2 methanation, which usually operated at 300–350 °C.20–23 Cobalt or iron based catalysts have also been widely investigated for CO2 methanation, and satisfactory performances were generally obtained at 400 °C or higher.24–28CO2. Design of catalysts that can work effectively at lower temperature has received considerable attention.29 Although many efforts have been made, the progress was restricted to several noble metal catalysts, especially at a temperature below 200 °C.30–36 Obviously, low temperature methanation of CO2 over cheap metal catalysts is highly desirable. Herein we show that the amorphous Zr-doped Co–B–O catalyst can effectively accelerate the CO2 methanation at above 140 °C. Excellent activity was obtained at 180 °C, which is comparable to or even higher than those of some noble metal catalysts . But its catalytic activity was much lower than that of the Co–Zr0.1–B–O catalyst. Using the liquid phase reduction method, we also prepared the catalysts with other promoters . Most of them  were also more effective than the Co/Zr0.1Ox catalyst, but they were markedly less efficient than the Co–Zr0.1–B–O catalyst. In short, Co–Zr0.1–B–O was an outstanding catalyst for low temperature CO2 methanation.In our work, the promoting factor was as high as 12.3, which is remarkably higher than those of the reported catalysts. The Co/Zr0.1–B–O catalyst are given in 0.1–B–O catalyst had no discernible diffraction peak, which further confirmed that it was amorphous , (200), (220) and (311) planes of CoO, and the peak at 44.2° was ascribed to Co (111). The results of N2 adsorption test of the catalysts are shown in Fig. S1.0.1–B–O catalyst can be classified as a type III curve, suggesting that multilayer adsorption occurred on the lyophobic surface. The Brunauer–Emmett–Teller (BET) surface area was 92.4 m2 g−1, indicating that it is not a porous material. The results of the EDS elemental mapping revealed that the Co, Zr, B, and O atoms were well dispersed in the catalyst , the reaction rate increased markedly and reached a maximum at 0.1. When the nZr/nCo was further increased the reaction activity decreased. This suggests that nZr/nCo = 0.1 was an appropriate ratio. The role of the Zr promoter in modulating the catalyst structure may be ascribed mainly to three aspects, i.e., changing the surface area of the catalyst, enriching the Co atoms on the catalyst surface, and varying the valence of the surface atoms. The BET surface areas and surface compositions of the Co–B–O catalyst and the Zr doped Co–B–O catalysts are given in Table S2.2 g−1) could be significantly enhanced by the Zr promoter. The surface area of the Co–Zr0.1–B–O catalyst was 92.4 m2 g−1, which was the highest surface area in the Zr doped catalysts. It is equally important that the Zr promoter could greatly enrich the active Co atoms on the catalyst surface. The Co content in total surface atoms of Co–B–O was 7.7%, while it reached above 30% in Co–Zr0.05–B–O and Co–Zr0.1–B–O catalysts. The synthesis of the Co–B–O catalyst is an exothermic process, which involves high surface energy and tends to cause agglomeration.38 The addition of the Zr promoter could affect the fabrication of the catalyst. Higher surface area and Co enrichment on the surface may cooperatively increase the active sites for the reaction. However, when excess Zr promoter was added, both the surface area and surface Co content of the catalyst decreased. This may explain partially why the performance of the Co–Zr0.3–B–O catalyst was not as good as that of the Co–Zr0.1–B–O catalyst. Besides the impact on the structure, the Zr promoter could also alter the valence of the surface atoms , and were much smaller than those of the Co–Zr0.1–B–O catalyst. This also helps to explain why the catalytic activity of the amorphous Co–Zr0.1–B–O catalyst was markedly higher than that of the Co/Zr0.1Ox catalyst fabricated by the commonly reported methods.To understand the impact of the peculiar structure on the reaction, we conducted the temperature programmed desorption (TPD) analysis, d H2-TPD . The res2 methanation, the undesired RWGS reaction also occurred, especially at relatively high temperature (>300 °C). The reaction route of CO2 methanation also depends on the composition and structure of the catalyst.42,43 In this work, no CO was observed under all conditions. Moreover, CO hydrogenation could hardly take place over the Co–Zr0.1–B–O catalyst, and the very small amount of CO consumed in the reaction was mostly converted to CO2 (Table S42 + C) occurred in the reaction. The carbon deposit generated in situ blocked the active sites and inhibited further reaction.44 To study the impact of the reactants on the valence of the catalyst, we conducted XPS characterization of the Co–Zr0.1–B–O catalysts after H2 adsorption and subsequent CO2 adsorption at reaction temperature  analysis of CO2 adsorption on the catalyst pretreated with H2 at 180 °C  and methoxyl  adsorbed on the catalyst surface.45 Several remarkable peaks between 2000 and 2150 cm−1 were also observed, which could be ascribed to the intermediates HxCOy  formed by H2, CO2 and/or hydroxyl groups on the catalyst surface.46 All these intermediates adsorbed weakly on the catalyst surface and disappeared immediately when the CO2 flow was stopped and H2 was introduced again. The in situ FTIR spectrum of the catalyst with simultaneous introduction of CO2 and H2 is given in Fig. S14.2 methanation conditions. The major peaks  are ascribed to the intrinsic feature of the catalyst evolved at the reaction temperature  were also observed by XPS characterization of the catalyst after CO2 methanation  containing carbonyl. Under optimized conditions, reactive intermediates were generated and transformed very quickly, accounting for the very high catalytic activity. The outstanding performance of the catalyst may originate from its strong ability to adsorb both reactants and the synergy of the atoms on the catalyst surface in converting them.Based on above discussion, we proposed the possible reaction mechanism. The CO0.1–B–O catalyst for CO2 methanation. The catalyst was very active and selective, and the activity of the catalyst reached 10.7 mmol2CO gcat−1 h−1 at 180 °C with a methane selectivity of 97.8%. The promoting factor of the Co–Zr0.1–B–O catalyst was as high as 12.3, which is remarkably higher than those of the reported catalysts. It is noteworthy that the catalytic performance is comparable to or even higher than that of some noble metal catalysts under similar conditions. The outstanding performance of the catalyst originated from two aspects. Firstly, the amorphous nature of the catalyst may lead to abundant surface defects and intrinsic active sites. Secondly, the Zr promoter could increase the surface area of the catalyst, enrich the Co atoms on the catalyst surface, and tune the valence state of the atoms at the catalyst surface. All these factors may enhance the activity of the catalyst. In the reaction, CO2 was reduced by the H atoms to methane via a series of intermediates, such as HCO3−, HCOO−, HCOOH, CH3O−, and other possible species HxCOy  containing carbonyl. We believe that this cheap and highly efficient catalyst has promising potential applications, and the protocol to design amorphous catalysts with promoters is useful to prepare other efficient catalysts using cheap metals.In summary, we report an amorphous Co–Zr2 (≥99.99%) and H2 (≥99.99%) were purchased from Sinopharm Chemical Reagent Co., Ltd (SCR). Zirconium nitrate pentahydrate (98%+) was provided by Adamas Reagent, Ltd. Cerium(iii) nitrate hexahydrate (99.5%) was offered by J&K, aluminum nitrate nonahydrate (99.99%) and chromium(iii) nitrate nonahydrate (99.95%) were bought from Aladdin.Sodium borohydride (98.0%), cobalt acetate tetrahydrate (99.5%), zinc nitrate hexahydrate (99.0%), CO0.1–B–O was obtained after centrifuging, washing with distilled water (200 mL), ethanol (30 mL) and acetone (30 mL), drying overnight under vacuum at 80 °C. Co–Zrx–B–O with different Zr amounts  was synthesized by a similar method. Zr–B–O and Co–B–O were synthesized using the above steps except for the absence of cobalt acetate tetrahydrate and zirconium nitrate, respectively.Metal-doped Co–B–O catalysts were prepared through the liquid phase reduction. Typically, solution A was first prepared by dissolving 0.303 g sodium borohydride in 10 mL distilled water containing 0.5 mL ammonia. 2 mmol cobalt acetate tetrahydrate and 0.2 mmol metal salts  were dissolved in a mixed solution of 20 mL distilled water and 5 mL ethanol to prepare solution B. Then solution A was added into solution B quickly at room temperature under vigorous stirring. The mixture was kept stirring for 20 min. Then the catalyst denoted as Co–M0.1Ox was synthesized by a coprecipitation method. 2 mmol cobalt acetate tetrahydrate and 0.2 mmol zirconium nitrate were dissolved in a mixed solution of 20 mL distilled water and 5 mL ethanol, then 10 mL of sodium hydroxide solution (0.8 mol mL−1) was added dropwise into the above solution under stirring, and after stirring for another 20 min a dark blue precipitate was obtained by filtration, followed by washing with 200 mL distilled water and drying overnight at 80 °C. The Co/Zr0.1Ox was obtained by calcination of the precipitate in air at 400 °C for 3 h followed by reduction with 5% H2 in Ar at 400 °C for 1 h.Co/Zr2 adsorption–desorption isotherms were recorded at 77 K using an ASAP2020 . The catalysts were treated under vacuum at 150 °C for 4 h before the test. The specific surface area was obtained by the Brunauer–Emmett–Teller (BET) method. XRD patterns of the catalysts were obtained using a Rigaku Ultima IV X-ray diffractometer with Cu Kα radiation (λ = 1.5418 Å) at a rate of 20.0° min−1 over the range of 20–90°. The X-ray photoelectron spectroscopy (XPS) experiment was conducted on an AXIS SUPRA (Kratos Corp.) with an Al Kα excitation source and all binding energies were referenced to the C 1s at 284.8 eV. High resolution transmission electron microscopy (HRTEM) was conducted on a Tecnai G2 F30 FETEM (FEI Corp.). HAADF-STEM images and EDS mapping were obtained using a Tecnai G2 F20 (FEI Corp). FT-IR spectra were collected using an NEXUS670 Fourier transform infrared spectrometer .The N2-temperature programmed desorption (TPD) was conducted using a Micromeritics AutoChem II chemisorption analyzer with He (30 mL min−1) as the carrier gas. About 0.1 g catalyst was charged into the quartz tube and heated to 100 °C at the rate of 20 °C min−1. After 1 h the temperature was cooled down to 50 °C, and CO2 adsorption proceeded with 10% CO2–90% He (v/v) mixed gas for 30 min with a flow rate of 50 mL min−1. Then the sample was purged with He of 50 mL min−1 for 1 h. Finally, CO2 desorption proceeded from 50 to 600 °C at 10 °C min−1. The H2-TPD measurement was performed using the same equipment with a similar procedure, except that 10% H2–90% He (v/v) was used.COIn situ FTIR spectra were recorded with a NICOLET iS50 FT-IR spectrometer  equipped with a high-temperature reaction chamber and a mercury cadmium telluride (MCT) detector at a resolution of 4 cm−1 and 32 scans per spectrum. The Co–Zr0.1–B–O catalyst and KBr were ground together and put into the sample cup. Then two tests were carried out using fresh catalyst, respectively. The first test was as follows: at 180 °C, the sample was first purged with N2 (100 mL min−1) for 2 h, and then it was treated with H2 (50 mL min−1) for 2 h. After further purging the sample with N2 (100 mL min−1) for 1 h, CO2 (100 mL min−1) was introduced and the CO2 adsorption began. Then the sample was further purged with N2 (100 mL min−1) for 2 h. In the end, it was treated with H2 (50 mL min−1) for 3 h. The background spectrum was recorded before CO2 adsorption. The second test was as follows: the chamber was first purged at 180 °C with N2 for 2 h and cooled down to 20 °C. After the CO2 was introduced for 5 min, both CO2 and H2 were charged and the sample was heated from 20 to 180 °C at the rate of 5 °C min−1 and kept at 180 °C for 0.5 h. The background spectrum was scanned before CO2 was introduced.2 was conducted in a 16 mL stainless-steel autoclave. Typically, 40 mg catalyst and 2 mL cyclohexane were added in the reactor. The reactor was sealed and the air in it was substituted with CO2 of 1 MPa three times, and then 4 MPa CO2 and 4 MPa H2 were charged successively at room temperature. The reactor was heated to 180 °C under stirring and was kept for 12 h. After the reaction, the reactor was cooled in an ice-water bath, and the residual gas was released slowly and collected for GC analysis (Agilent 7890A) equipped with a thermal conductivity detector (TCD) and flame ionization detector (FID). The amount of hydrocarbons such as methane was determined on the FID using a HP-AL/S column. The amount of carbon dioxide and hydrogen was obtained on the TCD using a HP-PLOT/Q column. The liquid phase was analyzed using an Agilent Technologies 7890B GC system with a flame ionization detector using a HP-5 column. The conversion of CO2 was the percentage of the CO2 charged into the reactor that was converted to hydrocarbon products, as is given below.The methanation of CO2 was the limiting reactant and it could more effectively reflect the proceeding of the reaction, calculation of the methane yield was based on the H2 charged into the reactor, as is shown below.Because H4 selectivity was the percentage of the C atoms in CH4 over the C atoms in total products, as is given below.The CH2 and H2) were charged to start the next run.After the reaction, the residual gas was released slowly. The gaseous and liquid samples were analyzed respectively. Then the reactor was sealed and fresh reactants (COThere are no conflicts to declare.SC-012-D0SC06414A-s001


The key role of hypoxia‐inducible factor 2alpha (HIF2α) in the process of renal cancer has been confirmed. In the field of tumour research, oxidative stress is also considered to be an important influencing factor. However, the relationship and biological benefits of oxidative stress and HIF2α in ccRCC remain unclear. This research attempts to explore the effect of oxidative stress on the cancer‐promoting effect of HIF2α in ccRCC and reveal its mechanism of action.The bioinformatics analysis for ccRCC is based on whole transcriptome sequencing and TCGA database. The detection of the expression level of related molecules is realised by western blot and PCR. The expression of Nucleoside diphosphate‐linked moiety X‐type motif 1 (NUDT1) was knocked down by lentiviral infection technology. The functional role of NUDT1 were further investigated by CCK8 assays, transwell assays and cell oxidative stress indicator detection. The exploration of related molecular mechanisms is realised by Luciferase assays and Chromatin immunoprecipitation (ChIP) assays.Molecular screening based on knockdown HIF2α sequencing data and oxidative stress related data sets showed that NUDT1 is considered to be an important molecule for the interaction of HIF2α with oxidative stress. Subsequent experimental results showed that NUDT1 can cooperate with HIF2α to promote the progression of ccRCC. And this biological effect was found to be caused by the oxidative stress regulated by NUDT1. Mechanistically, HIF2α transcription activates the expression of NUDT1, thereby inhibiting oxidative stress and promoting the progression of ccRCC.This research clarified a novel mechanism by which HIF2α stabilises sirtuin 3 (SIRT3) through direct transcriptional activation of NUDT1, thereby inhibiting oxidative stress to promote the development of ccRCC. It provided the possibility for the selection of new therapeutic targets for ccRCC and the study of combination medication regimens. 1. Silencing HIF2α in ccRCC can lead to a decrease in oxidative stress level.2. The oxidative stress pathway inhibited by the high expression of NUDT1 plays an important role in the cancer‐promoting of HIF2α in ccRCC.3. NUDT1 regulates ccRCC progression through cellular oxidative stress mediated by SIRT3 ubiquitination. DMEM  were used to culture cell lines, with 1% penicillin‐streptomycin solution and 10% foetal bovine serum  supplemented.Human ccRCC tissue samples come from Wuhan Union Hospital. The pathological results of all samples were ccRCC. All the patients had not been treated by any antitumour treatment before surgery. Huazhong University of Science and Technology Committee approved this study. The tissue samples were acquired with informed consent signed by patients.2.2The tumour and adjacent normal tissues of ccRCC patients were treated with paraffin embedding, and use immunohistochemical staining to process the tissue sections. The tissue sections were processed in sequence according to the following steps: deparaffinisation, rehydration and incubating for antigen retrieval. NUDT1 antibody  or HIF2α antibody  were used as primary antibodies and incubated overnight. Immune complexes and nuclei were visualised by DAB and haematoxylinm, respectively . The statistical analysis of all immunohistochemical staining is shown in Supplementary Information 2.3− ΔΔCT is used as the final statistical data. For ccRCC tissue samples, log 2(2 − ΔCT) is used as the final statistical data. Detailed primer sequences:GAPDH: forward, 5′‐GAGTCCACTGGCGTCTTCA‐3′,reverse, 5′‐GGTCATGAGTCCTTCCACGA‐3′;NUDT1: forward, 5′‐ATCGTGTTTGAGTTCGTGGG‐3′,reverse, 5′‐TGGAAACCAGTAGCTGTCGT‐3′;and HIF2α: forward 5′‐ACAGGTGGAGCTAACAGGAC‐3′,reverse, 5′‐CCGTGCACTTCATCCTCATG‐3′.TRIzol reagent  was used to extract total RNA from tissues. Use 1 μg RNA for reverse transcription. Determination of mass and concentration of RNA solution by NanoDrop2000 spectrophotometer . GAPDH was used as an internal control. Use SYBR Green Mix  to perform qPCR according to the manufacturer's instructions. Real‐time qPCR was performed using the StepOnePlus™ PCR system . Analyse qRT‐PCR data with StepOne software 2.3 . 22.46 cells were vigorously pipetted with 1 ml of TRIzol until clear. Oebiotech, China (contact NO: OE2017H0149S) performed total RNA extraction and total transcriptome sequencing after stable inhibition of HIF2α.HIF2α and NUDT1 stable knocked down ccRCC cell lines were established with lentivirus respectively. Every 5 × 10Total RNA extraction, whole transcriptome sequencing and bioinformatics data analysis after NUDT1 stable knockdown were supported by Majorbio (China). The differentially expressed genes from the RNA sequencing results were analysed via Majorbio Cloud Platform.2.5shNUDT1‐1 Forward 5′‐ccCGACGACAGCTACTGGTTT‐3′shNUDT1‐2 Forward 5′‐ccTGAGCTCATGGACGTGCAT‐3′shHIF2α‐1 Forward 5′‐caGTACCCAGACGGATTTCAA‐3′shHIF2α‐2 Forward 5′‐acTTCATGTCCATGCTGTGGC‐3′The supplier of HIF2α‐targeted shRNAs and NUDT1‐targeted shRNAs is Genechem Co. Ltd (China). The supplier of the overexpression plasmids of NUDT1 is Genechem Co. Ltd (China). The HIF1α siRNA was purchased from GenePharma. The shRNA or expression vector of NUDT1 was infected into 786‐0 and A498 cells with the manufacturer's protocols, respectively. The vector backbone of shRNA is ‘hU6‐MCS‐CBh‐gcGFP‐IRES‐puromycin’. The vector backbone of LV‐NUDT1 is ‘Ubi‐MCS‐3FLAG‐SV40‐EGFP‐IRES‐puromycin’. The specific sequences of shRNA were:2.6After the cells were lysed with RIPA, centrifuged at 13 000 rpm for 5 min, the supernatant was collected in two parts: a small amount of lysate was taken as input and the remaining lysate was incubated with 2 μg of the corresponding antibody and 30 μl of agarose beads overnight at 4°C. Immune complexes were separated at 3000 rpm for 5 min. Carefully discard the supernatant, and wash the agarose beads 3 times with 200μl of lysis buffer. Finally, add 64 μl of RIPA lysis buffer and 16 μl of loading buffer, and boil for 10 min.Western blotting: RIPA protein cleavage buffer  containing a mixture of protease inhibitors  and Phenylmethanesulfonyl fluoride (PMSF)  was used for protein extraction. The supplier of BCA kit is Beyotime Institute of Biotechnology . Forty micrograms of protein was loaded in to SDS‐PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes . Five percent non‐fat dried skim milk  was used to blocking. The membranes with primary antibodies were incubated overnight at 4°C. After washing with PBST 3 times, incubate with secondary antibody for 2 h at room temperature.The primary antibody was diluted at a ratio of 1:1000. The primary antibodies used were shown as follows: NUDT1 , HIF2α , GAPDH , HO‐1 , SOD2 , CAT , SIRT3 , Ubiquitin (P4D1) Mouse mAb  and OTUB1. The secondary antibodies used for western blotting were as follows: HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG(H+L) , HRP‐conjugated Affinipure Goat Anti‐Mouse IgG(H+L) . The above secondary antibodies are diluted at a ratio of 1:2000. The statistical analysis of all western blots is shown in Supplementary Information 2.73/ well. Cell proliferation rate was measured with cell counting kit 8  on the basis of the instruction manual. Add 110 μl CCK8 solution (10 μl CCK8:100 μl medium) to each well and incubate in the dark for 1 h. Determination of absorbance at 450 nm by NanoDrop 2000 spectrophotometer . Cell viability were measured at 0, 24, 48, 72 and 96 h after treatment.Each 96‐well plate was inoculated with cells at a density of 2 × 102.83 cells were seeded in 6‐well plates for 2 weeks and then fixed with methanol. Stain with 0.05% crystal violet  to visualise the colonies (>50 cells/colonies).A total of 102.9Ten microlitres pipette tips were used for wounding in a straight line when the cells have reached 70–80% fusion in the 6‐well plates. Afterwards, gently wash the cells with PBS and hold them at 37°C. Images were collected at 0, 12 and 24 h post wounding under UOP microscope  with UopView software .2.10® inserts  were used for migration and invasion assay. Cells are inoculated in the top compartment of the implant and allow cell invasion through the stroma. After 24 h of culture, fix the cells on the lower surface of the insert with methanol and stained with 0.05% crystal violet . Image acquisition and cell counting were performed in randomly selected areas.Cells are cultured in serum‐free medium for 24 h before testing. With or without Matrigel™  transwell2.115–106 cells/ml were measured for ROS levels through the Cellular ROS Detection Assay Kit . Fluorescence microscope  was used to observe the fluorescence intensity and collect images.Cells with a density of 5 × 10The assay uses ROS deep red dye to quantify ROS: the dye is cell permeable and reacts with intracellular ROS to produce a deep red fluorescent signal (Ex/Em = 650/675 nm). The intensity of deep red fluorescence reflects the level of cellular ROS; the stronger the fluorescence is, the higher the cellular ROS level is.2.12Use malondialdehyde (MDA) assay kit  to measure cell MDA. Intracellular malondialdehyde (MDA) expression was obtained at 532 nm.The protein carbonylation level was completed by the Protein Carbonylation Content Assay kit  according to the instructions.8‐oxodG levels in cells and tissues were determined by Human 8‐oxodG ELISA Kit  according to the instructions.2.13Respectively treat the cells with 50 μM cycloheximide (CHX) for 0, 4, 8, 12 and 24 h. Cells were collected for protein extraction for western blot analysis. ImageJ software was used to quantitatively analyse protein levels.Colchicine (S2284), chloroquine (S6999) and MG132 (S2619) were purchased from Selleck .2.143 cells in each well of 96‐well plate. After 24 h, 96‐well dishes were cultivated at 37°C for 1, 2 and 3 h for measuring the cell validity. The concentrations of sunitinib were 1, 2, 4, 8, 16 and 32 μmol/ml, respectivelyA498 and 786‐0 cells were suspended in DMEM  with sunitinib or DMSO. Inoculate 5 × 102.15® Kit (Agarose Beads) . The plasmids containing abridged NUDT1 promoter region . Construction vector is pGL3‐Basic. The specific construction sequences were shown in Figure 2.176 A498 cells infected with shNUDT1‐1, shNUDT1‐2 or negative control shRNA were subcutaneously injected into 6‐week‐old nude mice  and 4 × 106 A498 cells infected with negative control shRNA, shHIF2α, negative control shRNA+LvNUDT1 or shHIF2α+LvNUDT1 were injected into nude mice. The mice were euthanised 49 days after cell implantation and the tumour weight was measured. Measure tumour growth every week for 7 weeks with a digital caliper.A total of 2 × 102.186 A498 cells infected with negative control shRNA, shHIF2α, negative control shRNA+LvNUDT1, shHIF2α+LvNUDT1, shNUDT1‐1 or shNUDT1‐2 were injected into the tail vein of mice. All mice were euthanized, and the liver tissues were fixed, paraffin‐embedded and sectioned after 7 weeks’ observation. And then perform H&E and IHC staining. Use UopView software  to analyses the stained sections. The intensities of the staining were analysed using Image J.2.0 software .The metastatic ability of tumour cells was evaluated by the caudal vein metastasis model in nude mice. Note that, 1 × 102.19The Cancer Genome Atlas (TCGA) is a cancer genomics project containing data on more than 20 000 primary cancers. TCGA mainly stores basic information of various types of tumours, including RNAseq, miRNAseq, DNA methylation, patient clinical information etc. It is a relatively one of the public available comprehensive databases so far. The TCGA Kidney Clear Cell Carcinoma (TCGA‐KIRC) database contains clinical data and related gene expression data of 533 patients with ccRCC. The gene mRNA data in the bioinformatics analysis was obtained from the TCGA database. We analysed relevant data with SPSS 22.0 and generated the curves with GraphPad Prism 7.0.2.20https://www.oncomine.org). The mRNA levels of genes in normal renal and ccRCC tumour tissue and clinical information of ccRCC patients comes from the TCGA datasets .Screening was performed using three independent sets of genes associated with oxidative stress pathways from the Oncomine Database  to determining whether a set of genes defined a priori show statistical differences between the two biological states. It can be used to study the gene function and metabolic pathways of a group of statistically significant genes. We used GSEA v4.1.0 for Windows  to determine the enrichment pathway of the RNAseq data of ccRCC in TCGA database.2.21t‐test or analysis of variance with SPSS 22.0. Among them, independent‐samples t‐test is applied to test whether the mean and variance of the samples of two independent normal populations are from the same population; paired‐samples t‐test is applied to test whether two related samples come from a normal population with the same mean. The Pearson's correlation coefficient calculated by linear correlation analysis is used to calculate the correlation between the two genes. Receiver operating characteristic (ROC) and area under curve (AUC) are measured to obtain the highest overall accuracy to compare the diagnostic abilities of different genes.Statistical analysis adopts 33.1Oxidative stress is an important pathophysiological process of cells, which is closely related to the development of a variety of tumours. HIF2α is a key cancer‐promoting gene of ccRCC. Through bioinformatics analysis, we found that HIF2α is highly related with related to ccRCC's oxidative stress Figure . HoweverWe conducted further bioinformatics analysis to verify the above results. Similarly, the expression trend of NUDT1 in ccRCC was once again proved by data from the Oncomine database Figure . Further3.2The above studies have confirmed the characteristics of NUDT1 as a biomarker of ccRCC, so the specific biological function of NUDT1 in ccRCC is the focus of this part of the investigation. In order to analyse the biological functions of NUDT1, we used NUDT1 specific shRNA and overexpression lentivirus to construct ccRCC cell lines model with NUDT1 stably knocked down and overexpressed NUDT1 Figure . Analysi3.3CcRCC is a special tumour type that exhibits a significant change in cellular redox balance.3.4The above studies have confirmed that NUDT1 has a significant negative regulation of cellular oxidative stress. At the same time, cellular oxidative stress has been confirmed to play a vital role in tumour progression. Therefore, it is reasonable to believe that cellular oxidative stress also plays an important role in NUDT1's regulation of the progress of ccRCC. To verify the above hypothesis, we used acetylcysteine (NAC), an inhibitor of cellular ROS, to construct functional recovery models in ccRCC cells with stably NUDT1 knocked down Figure . As showEncouraged by the above results, the next focus is to explore the specific mechanism by which NUDT1 regulates cellular oxidative stress. As known, SIRT3 is an important cell oxidative stress regulator; especially it participates in the regulation of ROS in many tumour fields.3.5Considering the reasons of NUDT1 based on HIF2α knockdown and oxidative stress screening, as well as the important characteristics of NUDT1 as a cancer‐promoting gene, we have reason to believe that NUDT1 has a potentially key role in the cancer‐promoting pathway of HIF2α. We used NUDT1 overexpression lentivirus to construct functional recovery models in ccRCC cells with HIF2α stably knocked down to verify the above hypothesis Figure . As show3.62 to induce increased HIF2α levels. Compared with cells cultured under normoxia, the expression of HIF2α increased under hypoxic conditions, and NUDT1 increased accordingly . It is mainly located in mitochondria and is widely distributed in tissues and organs rich in mitochondria such as kidney, brain and liver. It can play a vital role in the deacetylation of histones and non‐histone proteins in the regulation of cell metabolism, cell cycle, cell apoptosis and cell lifespan.Many proteins have a dual role in tumours due to the differences in their localisation and functional pathways in cells. They play different roles in different stages of tumour development and in different cells, such as YB‐1,HIF2α as a far‐reaching ccRCC oncogene has been extensively studied in ccRCC. Most of these studies have focused on the angiogenic effects of HIF2α. Sunitinib, the first‐line treatment of ccRCC, targets vascular endothelial growth factor receptor (VEGFR) and platelet‐derived growth factor receptor (PDGFR), which are both downstream genes of HIF2α.5In summary, we found a novel pathway for HIF2α to transcriptionally activate the expression of NUDT1 in ccRCC. As HIF2α’s downstream, NUDT1 mediates the stability of SIRT3 to influence the process of cell oxidative stress and regulate ccRCC. Moreover, our research provides a new direction for the cancer‐promoting effect of HIF2α in ccRCC, which is different from angiogenesis. This may become a new weapon to break through the outcome of ccRCC‐targeted drug treatment resistance.The authors declare that they have no competing interests.Supplementary information 1Click here for additional data file.Supplementary information 2Click here for additional data file.Supplementary information 3Click here for additional data file.Supplementary information 4Click here for additional data file.


NMR spectroscopic data and molecular modeling calculations were used to ascertain the differences in binding mode and to shed light on the origin of recognition efficacy and selectivity.When facing the dilemma of following a preorganized or adaptive design approach in conceiving the architecture of new biomimetic receptors for carbohydrates, shape‐persistent macrocyclic structures were most often chosen to achieve effective recognition of neutral saccharides in water. In contrast, acyclic architectures have seldom been explored, even though potentially simpler and more easily accessible. In this work, comparison of the binding properties of two structurally related diaminocarbazolic receptors, featuring a macrocyclic and an acyclic tweezer‐shaped architecture, highlighted the advantages provided by the acyclic receptor in terms of selectivity in the recognition of 1,4‐disaccharides of biological interest. Selective recognition of GlcNAc Adaptive to be selective: Comparison of the binding properties of two structurally related biomimetic receptors toward disaccharides sheds light on the differences between a rigid macrocyclic architecture and a flexible tweezer‐shaped structure showing, counterintuitively, that the adaptive structure can be advantageous in terms of selectivity. NMR spectroscopy and molecular modeling calculations were used to provide a 3D description of the receptor‐disaccharide complexes in solution and explaining the origin of the observed selectivity. Lactose and maltose are two of the most common 1,4‐disaccharides, whereas cellulose and chitin, constituted by repeating units of cellobiose , and the α anomers of glucose, galactose, and fucose with affinities of 3.12, 1.19 and 360 μM, respectively.1 toward disaccharides were not yet explored. On the other hand, receptor 2, which has been shown to effectively recognize 1,4‐disaccharides, with a marked affinity (160 μM) and selectivity for the methyl β‐glycoside of GlcNAc2,We have recently reported two biomimetic receptors 2,1 effe1 toward the set of glucose‐containing disaccharides used to test receptor 2, to compare their binding properties and ascertain the effect of macrocyclic (preorganized) versus acyclic (adaptive) structures on recognition ability.In order to assess the role of the architecture in saccharide recognition, in this work we investigated the binding affinities of receptor 1H NMR spectroscopy, the binding ability of 1 was tested toward a set of disaccharides constituted by at least one glucose unit, for which the receptor showed good affinities, including cellobiose (CeB), lactose (Lac), maltose , trehalose (Tre), and sucrose .In a preliminary screening by 1, 1H NMR titrations of methyl‐β‐glycosides of cellobiose (MeβCeB), lactose (MeβLac), and maltose  were carried out in D2O (pD 7.4) at 298 K, additionally including MeβGlcNAc2, for which receptor 2 showed high affinity ,1H NMR titrations with MeβMal were also duplicated at pD 11 (Table S1) and fitted to the association model obtained at pD 7.4. While protonation of the aromatic amino groups is not expected in the investigated range of pD, the degree of protonation of the phosphonate groups does not affect the binding ability of receptor 1, as previously observed for binding to monosaccharidesFor a quantitative determination of the binding ability of 1 with disaccharides. Strong self‐association, with a dimerization constant of log βdim=3.84±0.20, favors complex species in which the receptor is dimeric, featuring two binding cavities and giving rise to complexes with stoichiometries higher than 1 : 1. Results show that receptor 1 effectively binds to MeβCeB, MeβMal, and MeβLac with good affinities, though with lack of selectivity, but does not recognize MeβGlcNAc2, for which no significant variations of chemical shifts were detected . Thus, receptor 1 can distinguish 1–4 from 1–1’ disaccharides (Suc/Tre), which are not bound at all, and MeβCeB from the N‐acetylated amino‐analogue MeβGlcNAc2, but cannot discriminate among glucose containing 1–4 disaccharides, proving to be insensitive to the configuration of the anomeric linkage  and to the presence of axial substituents (MeβLac). Surprisingly, these 1–4 disaccharides are bound with an affinity very close to that previously observed for MeβGlc, indicating lack of selectivity between mono‐ and disaccharides.As with monosaccharides, Table 1 and 2, as obtained by 1H NMR titrations, quantifies the selectivity advantage achieved through the adaptive architecture. Indeed, in contrast to 1, receptor 2 not only strongly binds to MeβGlcNAc2, but also discriminates among the investigated glucose containing 1,4‐disaccharides. Receptor 2 shows preference for the all‐equatorial MeβCeB, which is bound with an affinity very close to that observed for 1, whereas MeβMal and MeβLac are bound with an affinity more than one order of magnitude smaller.Comparison of binding properties between receptors 1 and 2, a description of the binding mode characterizing the receptor‐disaccharide complexes in solution was attempted by combining NMR techniques with molecular modeling calculations, following the approach previously adopted to study the complex between MeβGlcNAc2 and receptor 2, which provided an informative picture of the interaction.2 did not bind to 1, the investigation was carried out on MeβCeB, MeβMal and MeβLac.To shed light on the origin of such unexpected difference between 1, as calculated by nonlinear regression analysis of titration data, showed an upfield shift for all three disaccharides caused by the aromatic shielding effect  than on the H’‐1 proton (Δδ=0.24–0.32), suggesting a closer contact of the former to the aromatic moieties. On the other hand, from the analysis of chemical shift variations in the 1 : 1 complexes with receptor 2  for the H‐1 and H’‐1 protons of the latter are very similar, suggesting a fit of the entire disaccharide into the cleft of the receptor. The CSDs of MeβMal and MeβLac, significantly smaller and with the H‐1 CSD predominant, suggest a less comfortable fit in the cleft, in agreement with the corresponding lower affinities.Chemical shift variations of the anomeric protons H‐1 and H’‐1 upon formation of the 1 : 1 complex with receptor 1 and 2 with MeβCeB were then selected as representative examples, and their binding modes were studied by NOESY spectroscopy at pD 11, a medium in which the receptors are fully deprotonated species. From NOESY spectra run on the 1 : 1 mixture of 1 and MeβCeB, a strong intramolecular NOE contact was found between the H’‐1 and the H‐4 protons , suggesting that in the complex the disaccharide is in the conformation usually found in solution. Unambiguous intermolecular NOE contacts were also identified , the strongest of which were those between the OCH3 protons and both the H−C and H−D protons of the anthracene ring . The NOESY map shows a strong NOE cross peak between H‐2 and H−C, and a NOE contact of H’‐2 with the H−F located on the opposite side of the anthracene ring. Moreover, the OCH3 protons show NOE contacts with the H−C, H−D and H−E protons.NOESY spectra performed on an equimolar mixture of 1 with MeβCeB, on the assumption that, although prevalently dimeric, the receptor would feature two independent binding sites. A conformational search, using a well‐tested unconstrained molecular mechanics protocol,−1 from the global minimum that was in very good agreement with NMR spectroscopic data. The minimum energy structure depicted in Figure Based on NOESY NMR evidence, molecular mechanics calculations were carried out on the 1 : 1 complex of 2 featuring the N‐acetyl groups, can hardly fit into the receptor cavity. Thus, despite the good affinities observed, lack of selectivity between glucose containing 1,4‐disaccharides can be ascribed to the size of the macrocyclic cavity, unable to accommodate the entire disaccharide.The binding geometry obtained from calculations supports the observed affinities. Indeed, because the disaccharide is bound through the methyl glycoside unit exclusively, lack of selectivity among the investigated set of disaccharides can be easily anticipated, irrespective of the α/β glycosidic linkage to the second unit. This evidence also explains the closely similar affinities observed between the disaccharides and the monosaccharide MeβGlc. Likewise, the 1–1’ disaccharides, featuring a bulky substituent in place of the methyl group, and MeβGlcNAc2 and MeβCeB resulted in a single family of minimum energy conformers within 8.73 kJ mol−1 from the global minimum. The minimum energy structure depicted in Figure 2,The conformational search carried out on the 1 : 1 complex between 1, four hydrogen bonds were found between the diaminocarbazole unit and MeβCeB. However, in contrast to 1, a significant enhancement to binding could result from the extensive network CH‐π interactions that can be established between the axial protons of both the saccharidic units and the anthracenes (Table S6).Hydrogen bonding interactions could be calculated from the above model Figure d and, an2 can adapt to the disaccharidic guest better than the macrocyclic structure of 1, giving rise to increased affinity despite the lack of a hydrogen‐bonding unit. The evidence indicates that the latter is effectively compensated for by a tighter fit and by extensive CH‐π interactions. Such compensation is not fully achieved with MeβMal and MeβLac because axial substituents hamper a tight fit into the cleft, causing a drop in affinity. In contrast, the lack of preorganization and absence of a hydrogen‐bonding unit cause a severe drop in the affinity of 2 for monosaccharides, which is not compensated for by additional interactions; this results in undetectable binding. Thus, macrocyclic receptor 1 appears to be well preorganized for binding a monosaccharidic but not a disaccharidic guest, whereas acyclic receptor 2 can take advantage of its adaptive structure to establish more extensive attractive interactions with respect to its macrocyclic counterpart.The above three‐dimensional descriptions clearly show that the acyclic structure of 2 to MeβGlcNAc2 compared to MeβCeB could be explained by the additional hydrogen bonding and CH‐π interactions involving the N‐acetyl group that the former can establish with the receptor.The enhanced binding of 2 presents significant advantages over its macrocyclic counterpart 1, accommodating the disaccharidic guest within the binding cleft and selectively recognizing the methyl‐β‐glycoside of GlcNAc2 over a set of monosaccharides and structurally related 1,4 disaccharides. Because of its simple structure, easy synthetic availability, and potential for structural modifications, the tweezer‐shaped architecture of receptor 2 opens the way to the design of acyclic receptors for the recognition of saccharides in water.Together, the results presented demonstrate that a flexible acyclic structure can be an effective alternative to the widely studied macrocyclic architectures for the molecular recognition of neutral disaccharides in water, provided that a suitably designed combination of hydrogen bonding and CH‐π interactions can be established with the saccharidic guest. The structurally simple tweezer‐shaped receptor The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.