P1̄ space group. The second crystallizes into the uncommon dipole-parallel polar P1 space group, in which the molecular dipoles are aligned along a single axis and thus exhibit a high macroscopic polarization in its solid-state form. Due to the P1 polar packing, the sample can generate second harmonic light efficiently, about three times the intensity of the benchmark potassium dihydrogen phosphate. Our findings show that crystal engineering by hydrogen bonding in a single molecular backbone can be used for controlling the macroscopic NLO properties.Considering nearly infinite design possibilities, organic second harmonic generation (SHG) molecules are believed to have long-term promise. However, because of the tendency to form dipole-antiparallel crystals that lead to zero macroscopic polarization, it is difficult to design a nonlinear optical (NLO) material based on organic molecules. In this manuscript, we report a new molecule motif that can form asymmetric organic solids by controlling the degree of hydrogen bonding through protonation. A conjugated polar organic molecule was prepared with a triple bond connecting an electron-withdrawing pyridine ring and an electron-donating thiophene ring. By controlling the degree of hydrogen bonding through protonation, two different crystal packing motifs are achieved. One crystallizes into the common dipole-antiparallel nonpolar  We switch the molecule from SHG inert to SHG active by controlling the degree of hydrogen bonding through protonation. Unfortunately, these molecules tend to exhibit zero macroscopic polarization in the solid-state because they tend to form energetically favourable dipole-antiparallel assemblies by dipole–dipole interactions. Thus, it is a great challenge to design an NLO-active organic molecule that exhibits a large nonlinearity on the molecular level as well as on the macroscopic level.13–15Efficient SHG organic molecules are usually designed based on π-conjugated systems, due to their high polarizability and delocalized electronic structure.16 utilizing Langmuir–Blodgett (LB) film with asymmetry surfaces,17 and applying an external field to pole the material from symmetric to asymmetric.18 Ionic crystals consisting of molecular cations and anions have also been used to create stable non-centrosymmetric crystals utilizing the formation of strong electrostatic coulombic interactions and various π–π interactions between cation and anion layers.2 However, the introduction of multiple intermolecular and intramolecular interactions will increase the molecular and synthetic complexity towards achieving the targeted ordering in the crystalline state.Crystal engineering through inductive intermolecular interactions to stabilize the dipole-parallel packing paves the way towards achieving a non-centrosymmetric molecular assembly. Several methods have been utilized to stabilize the dipole-parallel molecular alignment, such as using asymmetric porous host structures to trap molecules inside the channels,P1 non-centrosymmetric packing in the solid-state and exhibits a large SHG response. Further protonation leads to hydrogen bonds on both nitrogen atoms and pyridine, resulting in a packing order with inversion of symmetry that does not exhibit SHG.In this work, we designed and synthesized a polar organic molecule, which exhibit macroscopic polarization by controlling the degree of hydrogen bonding between the molecules. Our molecule contains a large dipole from an electron-deficient pyridine ring and electron-rich thiophene ring connected by a carbon–carbon triple bond. The hydrogen bonding acceptor nitrogen atom in the pyridine ring and the hydrogen bonding donor nitrogen atom in the amine group are deliberately installed at the two ends of the molecule . By enlaThe electron-density distribution of the neutral molecule was simulated by plotting its electrostatic potential (ESPs) map . The endtert-butyl((5-ethynylthiophene-2-yl)methyl)carbamate 4 and 3-fluoro-4-iodopyridine 5. After purification, the –NH2 group was recovered by cleaving the –Boc group, followed by protonation using dilute HBr acid to produce the final salt, TM1. Adding more HBr will lead to further protonation of pyridine, which can be observed by an obvious color change from white (TM1) to yellow (TM2). The structure of TM1 and TM2 was characterized using pXRD, NMR , and Fourier transform infrared (FTIR) measurements. Compared with the neutral compound 7, the chemical shift of the protons of –CH2 adjacent to the end nitrogen atom in TM1 and TM2 downfield, from 4.07 ppm to 4.31 ppm. The protons of residue water in DMSO are also shifted downfield (3.3 ppm in TM1 to 3.7 ppm in TM2) due to protonation to H3O+ by the residual free acid in TM2.TM1 was synthesized with an overall yield of 57% through a 5-step synthesis process . The cor3+ group becomes broad and moves to lower wavenumbers (3000 cm−1) compared with reported non-protonated –NH2 (>3400 cm−1), making those vibrations indistinguishable and overlapping with the stretching vibration of C–H bond at both pyridine and thiophene rings. The strong and sharp peak at 2206 cm−1 refers to the stretching vibration of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>C. The asymmetric and symmetric bending vibration absorbing peaks of N–H in –NH3+ groups in TM1 are observed at 1608 cm−1 and 1494 cm−1, respectively. The stretching vibrational frequency peaks of non-saturated double bonds  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N, CC) are also observable in the range of 1610–1500 cm−1. Peaks in the figure print region (1350–400 cm−1) correspond to both in-plane (1320–1027 cm−1) and out-of-plane (817 cm−1) bending vibrations of unsaturated C–H and the stretching vibration of C–F. Hydrogen bonding is much stronger in TM2 than in TM1, thus the stretching vibration of N–H in –NH3+ group moves to low wavenumbers with increased intensity and band broadening. Meanwhile, the peaks of bending vibration of N–H in TM2 move to high wavelength numbers to 1634 cm−1 (asymmetric bending) and 1508 cm−1 (symmetric bending).19,20 Hydrogen bonding plays an important role in determining the final crystal stacking, where suitable hydrogen bonding numbers and strength will lead to a stable energetically-unfavourable polar packing.Powder X-ray diffraction (pXRD) patterns of the as-synthesized powder samples of TM1 and TM2 are in good accordance with the simulated ones, suggesting the phase purity of the as-synthesized powder samples . FTIR waP1  crystal, a standard inorganic SHG crystal, with the same particle size (about 100–150 μm). We found that the SHG intensity of TM1 is about three times higher compared to that of KDP . The position of the fluorine atom has an influence on the overall stacking of the molecules inside the crystal, and correspondingly will influence the macroscopic polarization.The single crystal XRD analysis was insufficient to resolve the positions of the fluorine atoms as they are not fixed in a single position. There is a small amount of disorder of the F atom about the two ortho sites. As shown in the potential scan plots Fig. S6, the enecis and -trans conformations of the units in the supercell  simulations to elucidate this phenomenon. We used a 2 × 2 × 1 supercell to explore the position effects of fluorine atoms on the macroscopic packing and polarizations. All the sixteen possible alignments of TM1 crystals Fig. S9 are consl Fig. S9. The sma packing , we founIn conclusion, we have synthesized an organic molecule TM1 with a large molecular dipole moment by connecting a highly electron-withdrawing pyridine ring and the strongly electron-donating thiophene ring through a triple bond. Hydrogen bonding allows the molecule to align molecular dipoles in a single direction resulting in NLO activity. As a result, this material exhibits a strong second harmonic generation signal, about three times the intensity of the benchmark KDP, showing that this material is amongst the best of SHG organic molecules. This is in contrast to the crystals made with the control molecule TM2, which exhibited an inversion symmetry and correspondingly no second harmonic generation signal. Our findings not only provide a useful NLO material with efficient SHG response, high optical transparency, and excellent thermal stability, but also shed light on crystal engineering using hydrogen bonding to obtain NLO materials. The results are expected to be general to other molecules that undergo hydrogen bonding and therefore expand the scope of NLO organic materials.2180022) and TM2 (CCDC 2180023).†Experimental details, synthesis and NMR spectra, stability measurements, and DFT simulation details. Crystallographic information files of TM1 (CCDC R. Zhao synthesized the materials, grew the single crystals, carried out the structure characterization, and wrote the manuscript. T. Zhu did the DFT simulations. R. Zhao and S. Wang measured the SHG. C. J.-Wilkins, A. Najjarian, and S. Hoogland provided suggestions for the project. A. Lough resolved the single crystal structures. E. Sargent and D. Seferos directed this project. All the authors provided suggestions and comments on the manuscript.There are no conflicts to declare.SC-013-D2SC03994J-s001SC-013-D2SC03994J-s002


Noncovalent interactionsare essential in the formation and propertiesof a diverse range of hybrid materials. However, reliably identifyingthe noncovalent interactions in nanocrystalline materials remainschallenging using conventional methods such as X-ray diffraction andspectroscopy. Here, we demonstrate that accurate atomic positionsincluding hydrogen atoms can be determined using three-dimensionalelectron diffraction (3D ED), from which the entire range of noncovalentinteractions in a nanocrystalline aluminophosphate hybrid materialSCM-34 are directly visualized. The protonation states of both theinorganic and organic components in SCM-34 are determined from thehydrogen positions. All noncovalent interactions, including hydrogen-bonding,electrostatic, π–π stacking, and van der Waalsinteractions, are unambiguously identified, which provides detailedinsights into the formation of the material. The 3D ED data also allowus to distinguish different types of covalent bonds based on theirbond lengths and to identify an elongated terminal P=O π-bondcaused by noncovalent interactions. Our results show that 3D ED canbe a powerful tool for resolving detailed noncovalent interactionsin nanocrystalline materials. This can improve our understanding ofhybrid systems and guide the development of novel functional materials. The results can thereforebe ambiguous, and typically only partial noncovalent interactionscan be resolved even when different spectroscopy techniques are combined.Spectroscopy techniques such as nuclear magnetic resonance (NMR),infrared, ultraviolet–visible, and Raman spectroscopy are usedto characterize noncovalent interactions.27 which enables the understanding of how noncovalent interactionsaffect covalent bond lengths and angles.28 However, SCXRD requires relatively large (>5 × 5 ×5 μm3) and well-ordered crystalline materials. Growinglarge crystalssuitable for SCXRD can be challenging and time-consuming, especiallyfor crystals that are formed via relatively weak noncovalent interactions.These factors can complicate, or even prohibit structure determinationof such materials by SCXRD.Single-crystal X-ray diffraction (SCXRD) is mostly used to determinethree-dimensional (3D) atomic structures and resolve noncovalent interactionsin crystalline materials,29 The noncovalent interactions resolved from PXRD data are thereforeoften ambiguous, owing to the large number of restraints that arerequired in structure refinement.Alternatively, powder X-ray diffraction(PXRD) can be used to gainstructural insights from samples composed of small micron- or nanometer-sizedcrystals. However, structure determination by PXRD often requiresphase-pure samples, and peak overlapping in the one-dimensional patterncan make structure determination difficult.34 Single-crystal neutron diffraction (SCND) has been used for studyinghydrogen-bonding interactions in a wide range of materials.33 A drawback is that the crystal size required for SCND is even largerthan that needed for SCXRD.32 Hydrogenpositions can also be determined by neutron powder diffraction; however,it is limited to crystals with relatively small unit cells where peakoverlapping is not severe.33 In addition,sample deuteration is often required for neutron powder diffraction,which can be both time-consuming and chemically challenging.33Compared to X-rays, neutronsare scattered by the nuclei of theatoms and have a unique advantage in locating hydrogen positions.4 times stronger interactions and significantlylower radiation damage per event compared to X-rays.34 This enables the use of electron diffraction for the structuredetermination of nanocrystalline inorganic and organic materials toosmall for X-ray and neutron diffraction.35 Electrons are more sensitive toward hydrogen atoms relative to X-rays,facilitating the location of individual hydrogen atoms in organicand inorganic samples at a subatomic resolution.41 Three-dimensional electron diffraction (3D ED) data are collectedanalogously to SCXRD and SCND using continuous sample rotation, asdemonstrated in MicroED and cRED; the latter is implemented in thesoftware Instamatic.43 Alternatively, 3D ED data can be obtained by combining stepwiserotation with precession or beam tilt,45 or mergingmany still diffraction patterns in SerialED.46 Recently, the rapid structure determination of organic compoundsfrom nanocrystals was demonstrated using continuous rotation datacollection at time scales comparable to SCXRD.39 During the past years, it has been shown that 3D ED can provideaccurate positions of non-hydrogen atoms in MOFs and HOFs.48 The noncovalent interactions such as hydrogen bonding could be identifiedbased on the donor–acceptor distances. However, 3D ED wasmainly used together with PXRD, NMR, and/or density functional theory(DFT) to reveal the entire range of noncovalent interactions in polycrystallinematerials.51 For example, 3D ED was used in combination with NMRto reveal the hydrogen-bonding network in small molecular crystals.49 While 3D ED was used to determine the positionsof non-hydrogen atoms, NMR was used to then assign correct atom types,locate hydrogen atoms, and derive the protonation state.49 Noncovalent interactions involving hydrogenbonding and π-stacking form the basis of heterochiral supramolecularpolymerization, which could be resolved using 3D ED data combinedwith DFT calculations.51Electrons are scattered by both the electronsand nuclei of theatoms and have 10Here, weexclusively use 3D ED to determine the structure of ananocrystalline hybrid organic–aluminophosphate material SCM-34.We show that the entire range of noncovalent interactions involvedin material formation can be revealed based on the atomic positionsincluding hydrogens refined against the 3D ED data. The accurate atomicpositions provide important insights into the hydrogen-bonding, electrostatic,π–π stacking, and van der Waals interactions betweenthe inorganic and organic components of the hybrid material, as wellas the protonation state and the effect of noncovalent interactionson the bond length of the terminal P=O π-bond. We corroborateour results using PXRD and NMR to confirm the unit cell parameters,our structural model obtained from 3D ED data, and the protonationstate of the organic and inorganic components.Figure S1, Supporting Methods). The resultingcrystals have a plate-like morphology with the average dimensionsof 3.0 × 1.5 × 0.2 μm3 . cRED data were collected at room temperature fromnine crystals during a 2 h session on a JEOL JEM2100 transmissionelectron microscope (TEM) using the program Instamatic.43 The data were processed using XDS,52 suggesting a triclinic unit cell . Seven data sets were selected based on theirintensity consistency, which were subsequently merged to achieve anoverall completeness of 98.8% up to 0.75 Å resolution (S3).We synthesizedthe hybrid material SCM-34 using 1-(3-aminopropyl)imidazole(API) as the structure-directing agent under hydrothermal conditions positions were successfully resolved. Duringstructure refinement using SHELXL,54 14out of 16 symmetry-independent hydrogen atoms were located directlyfrom the strong peaks in difference Fourier maps (S4). These were then constrained by ideal geometry but allowed to refinethe X–H  distances. The exceptions being theC2–H3 and C3–H4 distances that were restrained to theideal hydrogen bond lengths from a neutron diffraction of 1.08 Åwith a σ of 0.02 Å.55 The refinedchemical composition is |(C6N3H13)2[P4Al2O18H6], and the refinement converged to R1 = 0.186 andwR2 = 0.447 (F2 >2.0σ(F2); The structure of SCM-34 was solved ab initiofrom the merged cREDdata using direct methods in SHELXTier maps 1 and S4.4Al(PO4)2(OH)and AlPO-CJ10.57 The chains in SCM-34 are builtof AlO4(OH)2 octahedrons (Al3+) andO=PO2(OH) tetrahedrons (P5+), arrangedalong the crystallographic a direction. The adjacentAlO4(OH)2 octahedrons are connected via shearingthe protonated O1 atoms (proton: H9) and are further bridged by theO=PO2(OH) tetrahedrons (Table S4), whilethe bond lengths between P and O in P–O–H, P–O–Al,and P=O are 1.560(9)–1.580(8) Å, 1.508(7)–1.517(6)Å, and 1.492(8)–1.540(7) Å, respectively (Table S4). In the API molecule, the C–Nbond lengths in the imidazole ring and the chain tail are 1.318(14)–1.382(15)and 1.455(12)–1.492(10) Å, respectively (Table S5). The C–C bond length in the imidazole ringis 1.332(13) Å, much shorter than those in the chain tail (1.486(13)–1.498(13)Å).The SCM-34 structure consists of inorganic aluminophosphate chainsinteracting with the organic API molecules 2. The alahedrons 1 and 2. ahedrons 1. In the2(OH)tetrahedrons interact via hydrogen bonds between their terminal P–OHand O=P groups. The distance between the donor (D) and acceptor(A) atoms (P–O7–H11···O5=P) is2.554(10) Å, indicating that a strong interaction is formed tostabilize the chains. Meanwhile, the aluminophosphate chains are furtheraligned via strong hydrogen-bonding interactions (P–O4–H10···O9=P)between their neighboring parallel chains (4Al2O18H6]4–) withina single unit cell has a negative charge of −4, which is balancedby the positive charge from the two double-protonated API molecules(|(C6N3H13)2|4+). The protonated parts (−(NH3)+, −(NH)+−) of the API molecules are approaching the negativelycharged chains, binding to the chains along the b and c axes through hydrogen-bonding and electrostaticinteractions (31 The −(NH3)+ part interacts via three moderately strong hydrogenbonds  to the aluminophosphate chain.The −(NH)+– part has a strong hydrogen-bondinginteraction (N1–H1···O5). The API moleculesare observed to be packed as dimers via the imidazole rings with theshortest distance of 3.47 Å and a central distance of 3.75 Å,indicating the formation of the offset-type π–πstacking interaction.58 In addition, thevan der Waals interactions can be visualized by the Hirshfeld surfacefor the structure . The maximumdeviations of the unit cell lengths and angles are 0.22 Å and0.14°, respectively. The structural model of SCM-34 was validatedusing 31P, 27Al, and 13C NMR spectroscopy,inductively coupled plasma (ICP), and chemical element analysis. The 31P and 27Al NMR spectra indicate that P and Alare four- and six-coordinated, respectively . The 13C NMR spectrum shows that the API moleculesare accommodated in the structure and could be double-protonated . The calculated molar ratios of P/Aland C/N in SCM-34 are 2.1 and 2.0, respectively, which is consistentwith their molar ratios in the chemical composition (|(C6N3H13)2|[P4Al2O18H6]) resolved from the cRED data. 1H solid-state NMR and Fourier transform infrared (FT-IR) spectroscopieswere applied to detect the protonation state of the API moleculesand aluminophosphate chains. The 1H solid-state NMR spectrumshows a broad peak centered at 6.78 ppm and a sharp peak at 0.86 ppm. The sharp peak at 0.86 ppmwas assigned to Al–OH or P–OH groups;60 the broad peak (6.78 ppm), however, could not offer anyinformation regarding the protonation state of the API molecules.In the FT-IR spectrum, the signal attributed to Al–OH and P–OHgroups is overlapping at 3657 cm–1, and the signalof different C–H and N–H groups is overlapping in theregion of 2750–3200 cm–1 .61 The signal overlappingin the 1H solid-state NMR and FT-IR spectra makes it challengingto reliably interpret the protonation state of the API molecules andthe aluminophosphate chains without accurate structure information.The thermal stability of SCM-34 was studied by ex situ PXRD, in situelectron diffraction, and thermogravimetric analysis–differentialscanning calorimetry (TGA–DSC). The ex situ PXRD experimentsshow that the structure was stable up to 150 °C . In situ electron diffraction experimentsshow that the structure of SCM-34 was stable up to 100 °C andcollapsed at 150 °C in vacuum . TGA–DSC analysis indicates that the structure collapsedbefore 185 °C .To corroborateour results, we performed additional characterizationsthat were only used for structure validation. The unit cell dimensionsderived from the PXRD data are in good agreement with those determinedfrom our cRED data (Table S6).55 This is in line with previous observations that the hydrogenbond lengths observed in electron diffraction are closer to the inter-nucleidistances observed by neutron diffraction.62 The bond lengths of C2–H3 and C3–H4 had to be restrainedas the positions of H3 and H4 atoms were poorly resolved in our electrostaticpotential map (S4). This may be because H3 and H4are located in a region of the API molecule that has higher structuralflexibility and due to the fact that the two hydrogen atoms likelyare more disordered as they are facing outward the empty pockets betweenthe chains . The protons H1, H8C, and H9 in the structure indicatethat the aluminophosphate chains and the API molecules were protonatedduring the synthesis.We present the structure determination ofthe nanocrystalline hybridmaterial SCM-34. All atomic positions were resolved from our 3D EDdata, including the hydrogen atoms 1. The X–tial map 1 and S4.e chains 2. The hyTables S4 and S5). In theAPI molecules, the bond lengths of N1–C1, N1–C2, N2–C4,N3–C6, C2–C3, and C5–C6 are almost identicalto their corresponding reported SCXRD bond lengths (0.003 Å deviation).The C–N and C–C bonds in the imidazole ring and thetail of the API molecule can be distinguished based on their bondlengths (Table S5). In the aluminophosphatechains, based on the observed bond lengths, we can distinguish Al–Obonds in Al–O–Al (1.853(6)–1.864(4) Å) andAl–O–P (1.885(7)–1.930(6) Å) (Table S4). The different bond lengths betweenP and O in P–O–H, P–O–Al, and P=Ocan be identified from our data, with the exception of the P1=O5terminal bond length (1.540(7) Å; Table S4). Notably, the bond length of P1=O5 (1.540(7) Å) issignificantly longer compared to the expected bond length (1.500 Å)and the P2=O9 (1.492(8) Å) bond length. This elongationmay be the result of strong hydrogen-bonding and electrostatic interactionswith this oxygen atom (dO5···O7 = 2.554(10) Å in P1=O5···H11–O7and dO5···N1 = 2.586(10)Å in P=O5···H1–N1+).Systematic studies have shown the influence of strong hydrogen-bondinginteractions on the lengthening of terminal C=O bonds.28 The electron of the P1=O5 π-bondcan be delocalized by the hydrogen-bonding interaction, and the formalcharge of the O5 atom accordingly becomes −1.64 The strong electrostatic interactions between O5 and N1+ therefore may contribute to elongation of the terminal bond P1=O5.The shorter bond length of P2=O9 may be the result of weakerhydrogen-bonding and electrostatic interactions .The covalent bond lengths between thenon-hydrogen atoms in ourstructure are accurate with an average deviation of 0.013 Å comparedto the reported SCXRD bond lengths, enabling the assignment of eachbonding type  demonstrate the electrostatic interactions betweenthe positively charged API molecules and the negatively charged aluminophosphatechains. The API molecules are packed as dimers via offset-type π–πstacking interactions between the imidazole rings. Furthermore, vander Waals interactions between the chains and API molecules can bevisualized from the Hirshfeld surface.59 These noncovalent interactions can be calculated based on the accuratestructural model, which enables the quantitative analysis of noncovalentinteractions.66The hydrogen bond interactions were resolved basedon the identifiedpositions of the hydrogen, donor, and acceptor atoms in the structure.The strength of each interaction was interpreted based on the distancesof H···A and D···A and the angles ofD–H···A 2.65 The 4(OH)2 octahedrons and O=PO2(OH) tetrahedronsthat are stabilized via hydrogen-bonding interactions inside the chain.The resulting chains are further aligned with the hydrogen-bondinginteractions between their neighboring parallel chains. To extendthe structure into three dimensions along the b and c directions, API molecules are double-protonated and packedas dimers via π–π stacking interactions to placethe hydrogen-bonding and electrostatic interactions with the parallelaligned aluminophosphate chains and build the organic–inorganichybrid system. Van der Waals interactions play a role in shaping andsupporting the hybrid structure.Identifying the entirerange of noncovalent interactions in SCM-34enables us to deduce the formation mechanism of this organic and inorganichybrid material. Each aluminophosphate chain is built of AlO68 Being able to determine the detailed structures of nanocrystallinematerials, 3D ED can complement SCXRD and reveal the entire rangeof noncovalent interactions from nanocrystals. This method can alsobe applied for studying noncovalent interactions in other hybrid andnonhybrid materials such as hydrogen-bonded organic frameworks (HOFs)and covalent organic frameworks (COFs).69 We anticipate3D ED to provide new insights into the formation mechanism of crystallinematerials constructed via noncovalent interactions to improve ourunderstanding of supramolecular chemistry in polycrystalline materialsand facilitate the development of novel functional materials.The structure of the aluminophosphatechain in SCM-34 is similarto those reported previously in hybrid aluminophosphate materials,which were all determined by SCXRD from much larger crystals.


SbER10_X1 gene was used as a probe to identify ER family genes on the Setaria italica genomes (SiERs), and determine the characteristics of the SiERs family. Herein, the structural features, expression patterns, and thermotolerance of SiERs function were identified by bioinformatics analysis, real-time PCR and transgenesis estimation. Results showed that SiERs had four members: two members were located on chromosome 1 with a total of six copies , and two were on chromosome 4, namely, SiER4 (SiER4_X1 and SiER4_X2) and SiERL1. Among them, SiER1_X4 and SiER4_X1 were expressed highest in above-ground organs of foxtail millet, and actively responded to treatments with abscisic acid, brassinolide, gibberellin, and indole acetic acid. After overexpression of SiER1_X4 and SiER4_X1 in Arabidopsis, the plant height and biomass of the transgenic Arabidopsis significantly increased. Following high-temperature treatment, transgenic seedlings survived better compared to wild type. Transgenic lines showed higher SOD and POD activities, and expression level of AtHSF1 and AtBl1 genes significantly increased. These results indicated that SiER1_X4 and SiER4_X1 played important regulatory roles in plant growth and thermotolerance. The two genes provide potential targets for conventional breeding or biotechnological intervention to improve the biomass of forage grass and thermotolerance of field crops.Foxtail millet is commonly used as a food and forage grass. ERECTA (ER) is a receptor-like kinase that can improve plant biomass and stress resistance. The sorghum  Overexpficiency . When thenhanced . Furtherbidopsis . Overexpolerance . Interfe (PcBMM) . These rSiER family members (SiER s) in the foxtail millet were analyzed. SiER1_X4, and SiER4_X1 genes were isolated, and their biomass and thermotolerance of transgenic Arabidopsis were evaluated. The findings provide the functional genes for potential use in improvement of production and stress resistance in gramineous crops.In the current research, the characteristics of SiER gene tags from foxtail millet (Seita.4G086700.1 and Seita.1G338900.1) were obtained with the sorghum SbER10_X1 gene (XM_002437978.2) as a reference sequence after BLAST in the Phytozome v12.1 database. Four families of SiER members were obtained by searching the NCBI database with the two SiER tags to predict the complete CDS and chromosome-position information. The exon distribution (GSDS 2.0), cis-regulatory elements of promoters (Plant CARE), subcellular localization characteristics (Plant-mPLoc) and motif structure (MEME) of SiERs family were predicted. Moreover, the conserved functional domains (PROSITE and SMART databases), amino acid size, molecular weight, and isoelectric point (ProtParam) of SiERs proteins were analyzed. Two SiER1_X4 gene was listed in Based on the functional domains of SiERs, the amino acid sequences of the published ER family in monocot and dicot plants with similarity above 80% were downloaded from NCBI database , and cDNA was synthesized with a PrimeScript First-Strand cDNA Synthesis Kit . Taking the pedicel cDNA as material, specific primers  was as follows: 25 µL of 2 × PCR buffer, 10 µL of dNTP (2 mM), 1.5 µL of Primer-F (10 µM), 1.5 µL of Primer-R (10 µM), 1 µL of KOD FX , 5 µL of cDNA as template, 6 µL of ddH2O. The PCR procedure was as follows: 94 °C for 2 min, 40 cycles , and 68 °C for 10 min.Due to the abundant transcription of SiER1_X4 and SiER4_X1 (without the stop codon) was separated through SiER1_X4-gfpF1/SiER1_X4-gfpR1 and SiER4_X1-gfpF1 /SiER4_X1-gfpR1 primers , which included CaMV35S promoter. pJIT16318-SiER1_X4 and pJIT16318-SiER4_X1 were transferred into wheat mesophyll protoplasts (isolation from 10-day-old wheat seedlings) via the PEG4000-mediated method (The code fragment of  primers . The samd method . The traSiER1_X4 and SiER4_X1 segments (without the stop codon for fusion-protein development) were separated by primers of SiER1_X4-1302F1/SiER1_X4-1302R1 and SiER4_X1-1302F1/ SiER4_X1-1302R1, respectively  to obtain the fusion vectors of pCAMBIA1302-SbER1_X4 and pCAMBIA1302-SbER4_X1, respectively. Using a Agrobacterium tumefaciens-mediated transformation system (Arabidopsis (Columbia ecotype). The offspring seeds were screened with antibiotics to obtain homozygous transgenic SiER1_X4 and SiER4_X1 lines. The test steps were described by ectively . The samn system , the tar−2 s−1), five pots per transgenic line were treated in a light incubator at 42 °C for 48 h and 60 h, and the five remaining pots were cultivated at 26 °C for later biomass investigation (control).The stable transgenic lines overexpressing the target genes were cultivated on MS medium for 3 days (without antibiotics), and then moved into a light incubator for 7 days. Seedlings of the similar size were transplanted into pots (6.8 × 6.8 cm) with nine plants in each pot and ten pots per transgenic line. After 10 days of continuous growth in a greenhouse  lines were collected, and some samples were used to determine SOD and POD activity, as described by −2 s−1). After 6 days, mixture of the stems and leaves from a single plant for each variety was collected. After culturing the remaining plants for 15 days, seedlings were removed along with the roots, rinsed off the soil, and placed briefly on filter paper to dry, and then cultured in hormone solution or deionized water (control). The concentrations of the hormone solution were as follows: abscisic acid (ABA) 100 µM, brassinolides (BRs) 0.75 µM, gibberellin (GA3) 30 mM and indole acetic acid (IAA) 10 µM  were pre-germinated for 4 days. Seedlings with the similar germination were transplanted to pots (35 × 35 cm) with forty plants in each pot, and the flower pots were placed in a light incubator for growth  10 µM . SamplesSiERs expression patterns in diverse organs. Three individual plants were selected as biological replicates for each sample collection.In May 2021, the Dungu variety was planted in the experimental field, and embryo and coleoptile were collected at the germination stage. Roots, stems, flag leaves, flag leaf sheaths, pedicels, and inflorescence samples were collected at the flowering stage. Seeds were collected at the maturity stage. All samples were quickly frozen in liquid nitrogen after collection and stored at −80 °C for later detection of SiERs family were aligned to design specific primers for SiER1_X4 and SiER4_X1 qRT-PCR expression. The high-temperature related gene, AtHSFA1a, and superoxide suppressor gene, AtBl1, were used to determine the molecular-response mechanism of SiER1_X4 and SiER4_X1 in transgenic Arabidopsis plants after high-temperature stress (SiER1_X4 (SiER1_X4–qRTF2/SiER1_X4–qRTR2), SiER4_X1 (SiER4_X1–qRTF1/ SiER4_X1–qRTR1), AtHSFA1a (AtHSFA1a-qRTF2/AtHSFA1a-qRTR2), and AtBl1 (AtBI1-qRTF1/AtBI1-qRTR1), as well as the reference genes (SiActin -qRT F1/SiActin -qRT R1 and AtActin-qRTF5/ AtActin-qRTR5), are listed in −ΔΔCt method was used for quantification  and SiER4 (gene ID: LOC10175555 8097) were distributed on chromosome 4, and SiER1 was located on chromosome 1 with two genes (gene ID: LOC101780996 and gene ID: LOC117840131) (SiERL4 sequences (XM_004964364.4), and 2 copies and 27 exons in SiER4 sequences. In exon 25, 6 amino acids fewer were encoded in SiER4_X2 (XM_004964885.3) than in SiER4_X1 (XM_004964884.4). Three copies were found in the LOC101780996 gene of SiER1, exons 1 and 2 were lacking in SiER1_X3 (XM_014804622.2), 22 exons were found in the other two copies, 5 amino acids were lacking in exon 20 of SiER1_X2 (XM_014804623.2), and valine was lacking in exon 21 of SiER1_X1 (XM_014804625.2). Three copies were found in the LOC117840131 gene of SiER1, each of which contained 27 exons, compared with SiER1_X5 (XM_034720593.1), and mutations were found in exon 9 and 25 of SiER1_X4 (SiER1_X6 (XM_034720600.1). The amino acid structure prediction indicated that the proteins of the SiER4 family were larger, and the LOC101780996 of SiER1 was smaller. The nine copies of four genes in the SiERs family were all predicted to be transmembrane proteins, a typical feature of ER family proteins. In total 15 LRR tandem regions were detected in the SiERL4 protein, 13 LRR regions in SiER4, 9 LRR regions in LOC101780996 (SiER1), and 14 LRR regions in LOC117840131 (SiER1) (Four genes were found in the 7840131) . Further7840131)  showed tSiER1_X4 , and one (SiER1) .Arabidopsis AtER and AtERL. These findings showed that in the evolution of ER families of different species, ERL was a seperate branching direction, the phylogenetic relationship of SbER1 family was close to modern aquatic plants, whereas that of SbER4 family was closer to field xerophytic plants.In the published ER family, cluster analysis showed four categories : Categorcis-regulatory elements of SiERs family promoters were primarily involved in regulating three types of plant functional responses as follows (The  follows : (a) celThe SiERs is a typical receptor-like kinase , includiSiERs copies revealed the following  was larger, resulting in the largest sequence of SiER4 family. SiERL4 (LOC101753243) had 26 exons and was divided into a separate branch. It was reported that the ER family often constituted 27 exons, and ERL belonged to the ERECTA-LIKE1 family . With prolonged hormone-treatment time, the expression levels of the two genes showed a response pattern of initial increase and then decrease  treatment, the expression of SiER1_X4 slowly increased, whereas the expression of SiER4_X1 rapidly increased. At 6 h and 12 h respectively, the expression of the two genes reached their highest levels, respectively. Thus, compared with IAA treatment, the transcription level of the SiER4_X1 gene was higher under the other three treatments. These findings showed that SiERs actively respond to hormone induction and might participate in the regulation of millet development and stress-resistance related physiological processes.Upon treatments with the hormones abscisic acid, brassinolides, gibberellin, and indole acetic acid, decrease . After tSiER1_X4 and SiER4_X1 were 2973 bp and 2991 bp, respectively , respectively. Compared with WT, the plant height of the two transgenic lines significantly increased (P < 0.01), and the main stem diameter and the biomass per plant were significantly higher than those of WT lines (P < 0.05), indicating that overexpression of SiER1_X4 and SiER4_X1 gene could enhance the biomass of Arabidopsis. It had significant implications for improving the biomass of forage crops, such as sorghum and foxtail millet. Among them, the total number of siliques per plant of the SiER1_X4 transgenic lines was significantly more than those of WT lines (P < 0.05), whereas silique number was only slightly increased for SiER4_X1 lines. Meanwhile, plant height, total number of siliques per plant, and biomass per plant of SiER1_X4 transgenic Arabidopsis were higher than those of SiER4_X1 transgenic lines.stigated . In the Arabidopsis overexpressing SiER1_X4 and SiER4_X1 genes at elevated temperature (42 °C), the plant leaves withered and several plants showed local necrosis. After recovering for 11 days at 26 °C, only a few plants of WT lines showed vital signs, and the others all died; whereas the survival rate of transgenic Arabidopsis was extremely and significantly higher than that of WT (P < 0.01), expecially the SiER4_X1 transgenic plants, which showed a stronger ability to restore growth  (SiER4_X1 lines were slightly higher than those of SiER1_X4 lines.After treating e growth  and 10B. < 0.01) . Under hAtHSF1, and the superoxide suppressor gene, AtBl1, showed that the expression level of AtHSF1 in the transgenic lines was extremely and significantly higher than those of WT lines (P < 0.01) . Before and after high-temperature treatment, the AtHSF1 expression level of WT lines did not change significantly, whereas the expression level of AtBl1 significantly increased (P < 0.05). These findings suggested that overexpression of SiER1_X4 and SiER4_X1 genes may improve the high-temperature tolerance of Arabidopsis, which may be due to the influence of heat-related gene expression in the regulatory pathway and induction of the variable activity of related antioxidant enzymes. Moreover, SiER4_X1 showed a better regulatory function than SiER1_X4.Analysis of expression of the high-temperature regulation gene,  < 0.01) . ParticuSiER family members, two genes were distributed on the first chromosome, with a total of six copies, and two genes were distributed on the fourth chromosome, with three copies. In rice, wheat, sorghum, cotton, tobacco crops, the ER family also had two members, and each member had different spliceosomes, resulting in an uneven distribution of the number of introns and exons in the genome , and two copies on the fourth chromosome were closely related to terrestrial monocots. Further analysis of amino acid sequences of SiERs in other species showed that different ER families greatly differed in amino acid residues in the N-terminal signal-peptide recognition and transmembrane regions. ER family proteins are transmembrane proteins that can sense external stimuli, activate the expression of intracellular signal factors, and regulate the physiological response of cells  in foxtail millet. The foxtail-millet genome contained four SiERs member. Among them, SiER1_X4 and SiER4_X1 actively responded to the induced reaction of ABA, BRs, GA3, and IAA, with a higher expression level in above-ground organs of foxtail millet. Compared to wild type, the transgenic Arabidopsis lines overexpressing the two genes enhanced the plant height and biomass accumulation, and showed the higher SOD and POD activities under high temperature, reflecting an increased thermotolerance in Arabidopsis plants. These results provided potential targets for conventional breeding or biotechnological methods to improve forage crop production under harsh environments.This study analyzed the characteristics of 10.7717/peerj.14452/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14452/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14452/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14452/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14452/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.14452/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.14452/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.14452/supp-8Supplemental Information 8SiER1_X4 and SiER4_X1 expression patterns data. Overexpression of SiERs increased the biomass data of sorghum. Overexpression of SiERs enhanced the thermotolerance data of Arabidopsis.Click here for additional data file.


It is essential to see "when" and "where" 1O2 is produced and delivered to understand and utilize it. There is an increasing demand for molecular sensor tools to capture, store, and supply 1O2, controlled by light and engineered singlet and triplet states, indicating the 1O2-capturing-releasing state. Here, we demonstrate the outstanding potential of an aminocoumarin-methylanthracene-based electron donor–acceptor molecule (1). Spectroscopic measurements confirm the formation of an endoperoxide (1-O2) which is not strongly fluorescent and remarkably different from previously reported 1O2 sensor molecules. Moreover, the photoexcitation on the dye in 1-O2 triggers fluorescence enhancement by the oxidative rearrangement and a competing 1O2 release. The unique ability of 1 will pave the way for the spatially and temporally controlled utilization of 1O2 in various areas such as chemical reactions and phototherapies.Singlet oxygen ( Uncontrolled production of 1O2 causes undesirable degradation of materials and oxidative stress-induced disease progression. Therefore, the controlled and localized generation and sensing of 1O2 are essential and beneficial for utilizing 1O2 in chemical and biological reactions arbitrary.Singlet oxygen . Cycloaddition between 1O2 and a sensor affords an endoperoxide, blocking the PET and uncaging the emission5.1O2 sensing, the demand for capturing and controlled releasing of 1O2 has also been studied significantly in various areas in biology3 and chemistry4. However, it is challenging to produce it in the hypoxic tumor microenvironment3. This challenge is examined by stimuli-induced releasing of 1O216. Conventionally, 1O2 was liberated by heating endoperoxides of acenes or piperidones14. Fudickar et al. developed a dipyridylanthracene endoperoxide and released 1O2 under a chemical trigger13. Ucar et al. demonstrated a two-step chemical stimulus-induced release of 1O2 from naphthalene endoperoxide14. Phototrrigered 1O2 release is also reported by the photoexcitation on the anthracenyl part of the endoperoxide, although the high energy light of 282 nm laser was used16. Despite many 1O2 sensor molecules reported21, the sensor shows unexpected capture, store, and supply 1O2, indicating the capturing-releasing states with > 50-fold fluorescence intensity enhancement from the D-A form to the caged form and the endoperoxide.Apart from the 1O2 in a temporally controlled manner. It is identified that an aminocoumarin-methyl anthracene-linked molecule (1) traps 1O2 to form the endoperoxide (1-O2). Remarkably, 1-O2 is not as fluorescent as commercially available fluorogenic 1O2 probe molecules with an anthracenyl moiety. It is indicated in the present study that the unique molecular orbitals and triplet excitation energy levels of 1-O2 offer a weak fluorescent nature. An additional UV or NIR light-stimuli triggers the formation of a highly fluorescent compound. We also confirmed that 1-O2 releases 1O2 by photoexcitation on the dye molecule, coumarin, by one- or two-photon  excitation. The unique excited states originate from the aminomethyl anthracenyl moiety in 1-O2, making the efficient 1O2 capturing, storing, and releasing with fluorescence sensing achievable by one- or two-photon excitation. These unique phenomena are verified using spectroscopic measurements, including NMR and EPR, and density functional theory (DFT) calculations.Herein, the present study demonstrates the molecular dyad system that chemically traps, optically releases, and efficiently senses 2CO3), potassium iodide (KI), hydrochloric acid (HCl), and sodium azide (NaN3) were obtained from FUJIFILM Wako Pure Chemical Corporation, Japan. 7-Amino-4-methyl coumarin, 7-ethylamino-4-methyl coumarin, 9-chloromethyl anthracene, 9-methylanthracene, tetrakis(4-carboxyphenyl)porphyrin (TCPP), and Rose Bengal (RB) from Tokyo Chemical Industry (TCI), Japan. We obtained 2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) from Sigma Aldrich, USA. SOSG was obtained from Sigma and SiDMA sensor from DOJINDO, Japan. All the solvents were in reagent grade and from FUJIFILM Wako Pure Chemical Corporation, Japan.All chemicals used in this research were analytical grade and used as received unless otherwise stated. Potassium carbonate (KAbsorption spectra were recorded using an Evolution 220 UV–visible spectrophotometer (ThermoFisher Scientific), and fluorescence (FL) spectra were recorded using a Hitachi F-4500 FL spectrofluorometer. NMR measurements were performed using an Agilent Unity INOVA 500 or JEOL ECX-400 spectrometers. The continuous-wave-EPR measurements were carried out using a Bruker EMXplus spectrometer. For photoirradiation of samples, we used a DPSS 532 nm Green laser (Shanghai Dream Laser Technology), a Xenon/ Mercury lamp , or an 800 nm femtosecond laser . The 404 nm laser  with neutral density filters were used for varying the power.1 was prepared and characterized according to the reported procedure with slight modification20. 7-amino-4-methylcoumarin  and 9-chloromethylanthracene  were dissolved in 20 mL of acetonitrile. Then, DBU  was added to the solution, and the reaction mixture was stirred at 82 °C for 6 h. The reaction mixture was cooled to room temperature, and excess water was added, which provided a yellow residue. The pH of the solution was adjusted to ~ 6–7 using aq. HCl. The residue was filtered and dried. The yellow powder was re-dissolved in hot THF and then reprecipitated by adding an excess of toluene, and the residue was filtered and washed with toluene and then with acetone to give a pale-yellow powder . λmax (DMF): 354, 370, 389 nm. 1:1H NMR  δ = 8.50 (1H), 8.21 (2H), 8.05 (2 H), 7.57–7.40 , 6.74 (1H), 6.58 (1H), 6.02 , 5.21 (2H), 4.30 (1H), 2.40 (3H).2 was prepared according to the process as follows.2CO3  and potassium iodide  were added to the solution, and the reaction mixture was stirred at 85 °C for 5 h. The reaction mixture was cooled to room temperature, and excess water was added, which provided a yellow residue. The pH of the solution was adjusted to ~ 6–7 using aq. HCl. The residue was filtered and dried. The yellow powder was re-dissolved in hot THF and then reprecipitated by adding an excess of toluene, and the residue was filtered and washed with toluene and then with acetone to give a pale-yellow powder . λmax (DMF): 354, 370, 389 nm. 1H NMR  δ = 8.52 , 8.14–8.16 , 8.05–8.07 , 7.55–7.48 , 6.95–6.98 , 6.91–6.92 , 6.05 , 5.37 , 3.06–3.10 , 2.42 , 0.77–0.80 .7-(Ethylamino)-4-methylcoumarin  and 9-chloromethylanthracene  were dissolved in 10 mL of DMF. Then, K1 or 2; 10.0 μM) and a photosensitizer (5.00 μM) in DMF was photosensitized under selective photosensitizer excitation. The sample solution containing RB was irradiated with a 532 nm  continuous-wave laser. That containing TCPP was illuminated with a xenon lamp fitted with a 410–430 nm bandpass filter or a 404 nm  continuous wave laser. The sample solution was irradiated with a UV LED lamp (Asahi-spectra. Co. CL) . The FL and absorption spectra were recorded before and after irradiation.A sample solution of a sensor molecule (1 and 1.0 mM of RB were mixed in 800 μL of DMF (HPLC grade) and illuminated by a green diode laser . The reaction mixture was subjected to an HPLC system (Agilent 1220) equipped with C18-MS-II column  using DMF as the eluent. The fraction with the peak retention time of 2.8 min was collected and removed the solvent in a vacuum in the dark. DMSO-d6 was added and measured by an NMR spectrometer. Yield 86% (estimated from the HPLC profile). λmax (DMF): 354 nm, 1H NMR  δ = 7.53–7.56 , 7.49 , 7.31–7.33 , 6.98 , 6.92 , 6.86 , 6.47 , 5.98 , 4.50 , 2.35 .2.0 mM of -2, 10 min) and again measured by the NMR spectrometer. The results were shown in Figs The reaction mixture without the HPLC separation were also measured for comparison. And then, the sample solutions were irradiated with a UV LED lamp (Asahi-spectra. Co. CL) . The absorbed number of photons was calculated based on the induced light and absorbancy of the sample solution after the photosensitization of RB. The detected 1O2 in mole was calculated based on the used 1  and the signal change ratio of TEMP to be TEMPO (50%).The singlet oxygen photo-releasing quantum yield is estimated to be 1.6% based on the absorbed number of photons (320 nmol) and the detected 1 (10.0 μM) and a photosensitizer (5.00 μM) in DMF was photosensitized under selective photosensitizer excitation. The sample solution containing RB was irradiated with a 532 nm (50 mW) continuous-wave laser. Then, SOSG (10 μM) was added and the sample solution was irradiated with a UV LED lamp (Asahi-spectra. Co. CL) . Before adding SOSG, the solution was purged with argon  for the condition of the absence of oxygen. The FL and absorption spectra were recorded before and after irradiation.A sample solution of 1 (10.0 μM) and rose bengal (10.0 μM) in DMF was irradiated under a 532 nm green laser (50 mW) for the photosensitized generation of 1O2. The sample's FL and absorption spectra (250 μL in 5 mm pathlength cuvette) were recorded before and after 30 min of the photosensitization. Then, the sample solution was irradiated with an 800 nm fs laser (Coherent Mira 900) for 40 min, and FL and absorption spectra were recorded every 5 min interval. The samples' FL quantum yield was estimated by a relative FL quantum yield estimation using coumarin 120 as a reference. A control experiment was conducted to compare the enhancement factor by recording the FL spectra of the equivalent sample solution after photosensitization, which was kept under dark.A sample solution containing 1 or 2; 10.0 μM) and a photosensitizer (5.00 μM) in DMF was photosensitized under selective photosensitizer excitation. The sample solution containing RB was irradiated with a 532 nm  continuous-wave laser. That containing TCPP was illuminated with a xenon lamp fitted with a 410–430 nm bandpass filter or a 404 nm  continuous wave laser. The sample solution was irradiated with a UV LED lamp (Asahi-spectra. Co. CL) . The FL and absorption spectra were recorded before and after irradiation.A sample solution of a sensor molecule ” and “Density = ” keywords after performing TD-DFT calculations.The molecular structures and electron energies were optimized and obtained by the Gaussian16 package1O2 was indirectly monitored by using a spin probe TEMP that undergoes oxidation by 1O2 to form EPR-active TEMPO. The measurement conditions were optimized by evaluating the photosensitization of RB in the presence of TEMP. To this purpose, 5 mM of TEMP was added to 5.00 μM RB in DMF. The solution was irradiated with a xenon lamp fitted with > 480 nm long-pass filter for 30 min (50 mW at 532 nm). After the photosensitization, the EPR spectra of the sample solution were recorded using the X band frequency of microwave (9.79 GHz) at 1 mW cm−2 power. To check the possibility of generation 1O2 under UV illumination, 1 or RB was illuminated with a UV lamp with an emission maximum at 365 nm, at 2.0 mW cm−2 for 10 min in the presence of 5.00 mM of TEMP.The generation of 1O2, a sample solution containing 1 (10 μM) and RB (5 μM) was irradiated with a xenon lamp fitted with > 480 nm long-pass filter for 30 min (50 mW at 532 nm). After the photosensitization and generation of the intermediate complex, 5 mM of TEMP was added to the sample solution, and EPR spectra were recorded before and after 10 min of UV illumination  . A control experiment was conducted by illuminating a sample solution containing 1 (10 μM) and RB (5 μM) and 5 mM of TEMP with UV light .To examine the UV-activated release of 1 to be 100%   by green light to form a moderately fluorescent endoperoxide 1-O2  Fig. ” sectiona1 Fig. ”ty Figs.  and S7. 1-O2  also guarantee the feasibility of the formation of 1-O2 7 although the original dye part of 1-O2  is highly fluorescent (ϕ = 0.62)19. This result implies the huge fluorescence intensity enhancement possibility during 1O2 sensing and the existence of a non-radiative relaxation pathway in the photoexcitation of 1-O2, which is discussed below. Interestingly, a short-time illumination of UV light  on both the reaction crude between 1 and 1O2 or the isolated 1-O2 induced the remarkable fluorescence intensity enhancement  suggest that the UV illumination significantly accelerated the change in the molecular structure. Anthraquinone is one of the resultant products after UV illumination due to an oxidative rearrangement29, supported by the 1H-NMR spectrum of the isolated 1-O2 after UV illumination   analyses were studied  of the molecule. The calculations on 1-O2 predict the several triplet excited states (T2–T5), which may possess comparable energy levels to the S1 state  and HONTO of 1-O2 (S0 → T3)   Figs.  and S2. f 1 Fig. . These r1O236. Remarkably, the UV (365 nm) illumination triggers 1O2 release in parallel with the fluorescence intensity enhancement, as evidenced by the EPR results . Moreover, the same phenomena, the 1O2 detection only after the photosensitization of RB in the presence of 1, were also observed by using SOSG or SiDMA , at which anthracene endoperoxide does not possess optical absorption, than those in the previous reports showing the phototriggered release of 1O2 by the illumination (hv: 282 nm)16 on the anthracene moiety26. Furthermore, the release was also detected by two-photon excitation. The linked dye molecule of 1, coumarin, would change the usable wavelength of light for the phototriggered release from the endoperoxide. The enhancement factor of the EPR signal is ca. 50% relative to the control using TEMP and RB without 1  and the detected 1O2 (10.0 nmol × 50% = 5.00 nmol).It should be noted that the applied wavelength of light is significantly longer . Here, after photosensitization of RB in a sample solution containing 1 and RB with the 532 nm, we applied the NIR light. As a result, 1 showed a threefold enhancement in the fluorescence quantum efficiency after 40 min. This enhancement suggests the release of 1O2 by the two-photon absorption mediated photoactivation of 1-O2  during the reactions with 1O2. The fluorogenic 1O2 sensing ability of 1 is unlocked by supplying additional energy by low-intensity UV light or low-energy NIR light after the 1O2 trapping to form 1-O2. The intermediate 1-O2 is stable against 1O2 scavengers and high temperatures in the dark. Unlike the reported anthracene-linked molecules in the endoperoxide form, 1-O2 is rather nonfluorescent. We attributed the photo-triggered fluorescence intensity enhancement to the molecular rearrangement and unique intersystem crossing in 1-O2. A photo-triggered release of 1O2 was observed by UV/NIR light illumination on the dye molecule in 1-O2 with ~ 50% yield using EPR spectroscopy. DFT calculations support that the unique excited states and the molecular orbitals of 1-O2 offer temporal control over 1O2 capturing, storing, releasing, and sensing. The findings in the present study provide valuable information in the photoexcited state engineering to create novel photofunctional molecular sensors. It will also pave the way for the spatially and temporally controlled utilization of 1O2 in broad areas such as 1O2-mediated chemical reactions and photodynamic therapy.We revealed the unique properties of an anthracene-coumarin donor–acceptor system (Supplementary Information.


Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both  PPM1D locus or gain-of-function mutations in the last exon of PPM1D have been reported to promote tumorigenesis by inhibiting p53 pathway and are commonly found in various solid tumours and haematological malignancies , HEK293, human breast adenocarcinoma MCF7 or human osteosarcoma U2OS cells were grown in DMEM supplemented with 6% FBS (Gibco), Penicillin and Streptomycin. U2OS-PPM1D-KO cells with a knock-out of PPM1D were described previously . HeLa ceCoding sequence of human TRF2 was PCR amplified from pLPC-NMyc-TRF2 (Addgene ID: 16066)  and inseThe following antibodies were used in this study: TRF2 , TIN2  from Abcam; TRF2 , TIN2 , RAP1 , 53BP1  from Novus Biologicals; TRF2 , TIN2 , TPP1 , RAP1 , PPM1D , PPM1D  from Santa Cruz Biotechnology; Phospho-Histone H2A.X (Ser139) , KAP1-S824 (#4127) and PPM1D  from Cell Signaling Technology; γH2AX , GFP , FLAG , Fk2  from Roche. A custom-made pTRF2-S410 antibody was generated by immunization of rabbits with KLH-conjugated phospho-peptide RLVLEEDpSQSTEPSA corresponding to amino acids 403–417 of the human TRF2 (according to the numbering in reference sequence NP_005643.2) (Davids Biotechnologie). Subsequently, immune sera was affinity purified using negative and positive selection with non-phosphorylated and phosphorylated peptides, respectively. PPM1D inhibitor GSK2830371 was from MedChemExpress and was validated previously . Validat2, 300 mM Sucrose, 0.5% Triton X-100 for 5 min. After washing in PBS, coverslips were blocked with 1% BSA in PBS for 30 min, incubated with primary antibodies for 2 h at room temperature and subsequently with Alexa Fluor secondary antibodies (Thermo Scientific) for 1 h. After incubation with DAPI for 2 min, coverslips were washed with water and mounted with Vectashield. For proximity ligation assay (PLA), coverslips were stained with the indicated primary antibodies followed by incubation with PLA probes  for 1 h at 37 °C, ligation for 30 min at 37 °C, and polymerase reaction for 2 h at 37 °C according to the manufacturer's protocol . For immunofluorescence-FISH, coverslips were fixed, permeabilized, and blocked as described above. After dehydration with 70%, 95% and 100% ethanol for 3 min each, the coverslips were incubated for 10 min at 80°C face down on a slide with 20 μl of hybridization solution , and 0.5% blocking reagent . Hybridization was performed for 2 h at room temperature in a humidified chamber in dark. The coverslips were then washed twice for 10 min in wash buffer 1  and twice for 5 min in PBS. Incubation with primary antibodies was performed overnight at 4°C, followed with PBS wash and incubation with secondary antibodies for 1 h at room temperature. The coverslips were then stained with DAPI, rinsed in water and mounted using Vectashield. For the high content microscopy, images were acquired using Olympus ScanR equipped with 60×/1.42 OIL objective and analyzed using ScanR analysis software. Confocal imaging was performed using Leica DMi8 equipped with HC PL APO 63×/1.40 OIL CS2 objective. Images were acquired as Z-stacks of five planes with voxel size 44 × 44 × 129.7 nm and 3D-deconvolved using Huygens Professional (Scientific Volume Imaging) based on the theoretical point spread function. Metaphase spreads were imaged using Leica DM6000 equipped with a HCX PL APO 63×/1.40 OIL objective and a sCMOS Leica DFC 900 camera.Cells grown on coverslips were washed in PBS, fixed by 4% PFA for 15 min and permeabilized with 0.2% Triton-X100 for 5 min. Where indicated, cells were pre-extracted prior fixation in 25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM EDTA, MgCl6 cells/ml, and dropped onto frozen slides from distance of 30 cm. Slides were air dried overnight, washed 3 × 5 min in PBS and hybridisation was performed as described above. After washing in wash buffer 1 and three times 10 min in wash buffer 2 , slides were stained with DAPI, PBS washed, dehydrated with 70%–95%–100% ethanol series, and mounted in Vectashield.Cells were synchronised in late G2 phase by treatment with 9 μM R0-3306 (MedChemExpress) for 16 h. After washing with PBS, cells were released into media supplemented with 0.1 ug/ml colcemid (Sigma) and incubated for 3 h. Subsequently, cells were trypsinised, pelleted at 300 g for 5 min and resuspended in 5 ml of warm 75 mM KCl. After incubation for 30 min at 37°C, cell suspension was mixed with 1.25 ml of fixative solution  while vortexing. After centrifugation, cells were 3× washed with fixative solution. Finally, cells were resuspended in 200–800 μl of fixative solution to achieve concentration 4 × 102, 0.5 mM DTT, 0.5 mM PMSF) supplemented with cOmplete protease inhibitor cocktail (Roche). After 5 min of incubation, cells were pelleted at 500 g for 5 min at 4°C and resuspended in 2 volumes on NE buffer. Nuclei were released from cells using Dounce homogeniser and collected with centrifugation at 800 g for 5 min at 4°C. Nuclei were resuspended in nuclei wash (NW) buffer  in concentration 1–2 × 107 nuclei/ml, and incubated with 100 μg/ml of Trioxalen (Sigma) on ice while stirring in the dark for 5 min. Crosslinking was performed by exposing 2 ml of nuclei suspension at a 6-well plate to 365 nm light for 30 min on ice. Cross-linked nuclei were centrifuged at 800 g for 5 min at 4°C, washed with ice-cold NW buffer, and resuspended at 1 × 107 nuclei/ml. The nuclei suspension was diluted 10× in spreading buffer  pre-warmed at 37°C, and 100 μl of the suspension was immediately dispersed on 13 mm round 1.5H coverslips using cytospin at 600 rpm for 2 min. Coverslips were dried at room temperature for 1 h and fixed in methanol:acetic acid (3:1) for 1h. Coverslips were dehydrated with 70%–95%–100% ethanol series and hybridized with TelC-Cy5 PNA probe overnight at 4°C in a humidified chamber protected from light. After washing with wash buffer 1 and wash buffer 2, coverslips were washed in water, air-dried and mounted with Vectashield.For super-resolution imaging of telomeric loops, we used modified protocol from Doksani et al. Parental U2OS and PPM1D KO cells were trypsinized, washed with PBS and resuspended in 5 volumes of ice-cold nuclei extraction (NE) buffer  was performed using DeltaVision OMX™ V4 equipped with Blaze Module  and a PLAN APO N 60×/1.42 OIL objective. A 568 nm OPSL laser was used for excitation and a pco.edge 5.5 sCMOS camera for signal detection. Raw images were acquired in a z-stack with 125 nm step, 8 z slices, 15 images per slice, pixel size 80 nm. The image reconstruction was performed using SoftWorX software . Blinded analysis of telomeres in maximal projection images was done as previously described . Only teHEK293 stably transfected with empty pBIOID2 or pBIOID2-PPM1D-D314A were grown in media supplemented with 50 μM biotin for 5 h, then cells were collected, washed in cold PBS and lysed under denaturating conditions in lysis buffer . Protein lysates were diluted with four volumes of PBS and sonicated 3× for 30 s. Cell lysates were cleared by centrifugation at 15 000 g for 10 min and biotinylated proteins were pulled down by incubation with Dynabeads M-280 Streptavidin for 90 min. After washing twice in lysis buffer and twice with PBS, on-bead trypsin digestion was performed and peptides were analyzed by mass spectrometry using Orbitrap Fusion instrument . All data were analyzed and quantified using MaxQuant (version 1.6.2.1) and Perseus softwares . Three b2) supplemented with PhosSTOP and protease inhibitors (Roche), sonicated and DNA was digested by Bensonase. Cell extracts were incubated with GFP Trap beads (Chromotek) for 1 h and after washing, proteins were eluted by Laemli buffer and analyzed by immunoblotting.HEK293 cells were transfected with pEGFP, pEGFP-TRF2 or pEGFP-TRF1 and collected after 48 h. Cells were extracted by IP buffer  supplemented with PhosSTOP and protease inhibitors. Beads were washed with a phosphatase buffer and incubated with mock or with 150 ng of the purified His-PPM1D for 20 min at 37°C. Reaction was stopped by addition of 4× concentrated Laemli buffer.Expression and purification of human His-PPM1D was described previously . EGFP-TRBiotin-Ahx-ISRLVLEEDpSQSTEPSAGLN-amide (TRF2-pS410) and Biotin-Ahx-ISRLVLEEDSQSTEPSAGLN-amide (TRF2-CTRL) peptides were synthesized (Genscript), dissolved in ammonia water and then diluted to 1 mg/ml in TBST . Peptide pull down was performed as described . Dynabeaex 494 nm, λem 518 nm) were synthesized by GenScript. Peptides (3 nM) in a 1.5 ml quartz-glass cuvette with a magnetic stirrer were titrated with TIN2 to a final concentration of 500 nM in 50 mM NaCl in 50 mM phosphate buffer, pH 7.0 at 25°C. Fluorescence anisotropy change upon addition of TIN2 was measured at λex 490 nm, λem 520 nm with excitation and emission slits 9 nm. Fluorescence anisotropy was measured three times, and averaged with a relative standard deviation always lower than 3%. The value of the dissociation constant was determined by non-linear least square fits according to the equation: r = rMAXc / (KD + c) using OriginPro 2022 (OriginLab Corporation) . Only two-tailed test were used. Student's t-test were performed under the assumption of normality. As a nonparametric test, we used Mann–Whitney statistics. All experiments were reproduced with similar results at least two times.in vitro, suggesting that the interaction between TRF2 and PPM1D is direct (Protein phosphatase magnesium-dependent 1 delta PPM1D) is a chromatin-bound protein with poor solubility making analysis of its interacting partners a major challenge . To iden is a chrs direct .Finally, we tested the interaction between PPM1D and shelterin in cells using a proximity ligation assay . We obseTaken together, we conclude that PPM1D interacts through its catalytic domain with several components of the shelterin complex in various cell types regardless of the type of telomere maintenance, including telomerase proficient MCF7 cells and alternative telomere lengthening (ALT)-dependent U2OS cells.Apart from functions at telomeres, TRF2 and TRF1 were reported to localize also to other chromatin compartments . TherefoTo identify the regions in PPM1D that are necessary for its localization at telomeres, we transfected cells with plasmids expressing EGFP-tagged PPM1D or its deletion mutants. We found that the wild-type EGFP-PPM1D, a deletion mutant lacking the Proline-rich region (referred to as ΔPro) and a PPM1D-A380 mutant comprising of the catalytic domain between amino acids 1–380 all were enriched in TRF2 foci Figure . In contin vitro . Therefore, we generated an affinity purified rabbit polyclonal antibody against the phosphorylated S410 of TRF2 that is conserved across species, matches a consensus motif for ATM/ATR and PPM1D and has previously been detected in cells exposed to ionizing radiation or to treatment with cytarabine  64–66)..64–66). o Figure . Next, wo Figure . In non-o Figure . As expeo Figure ,44,49. Io Figure . To valio Figure . As expeo Figure . Furthero Figure . We concAs the basal level of pTRF2-S410 signal in non-treated cells was relatively low, we searched for conditions that would stimulate the phosphorylation of TRF2. Upon exposure to ionizing radiation, DSBs are randomly generated across the genome making interpretation of events observed at telomeres difficult. To induce DSBs specifically at telomeres, we transfected cells with a plasmid expressing Cas9 and a sgRNA targeting the telomeric repeats . ConsistFinally, we induced DSBs at telomeres in cells treated with small molecule inhibitors of the major protein kinases responding to DNA damage and assayed the impact on protein phosphorylation at telomeres. Similarly to DSBs induced by TRF1-FokI, we observed that inhibition of ATM reduced the level of γH2AX at telomere breaks induced by Cas9 Figure  70). In. In70). TRFH (residues 58–268) and TRF2 TRFH (residues 84–287) domains interact with a TRFH-binding motif (TBM) of TIN2 (residues 256–276)  lacking the S410 site , pulled down the TIN2-TPP1-POT1 trimer from the nuclear extract. Subsequently, a fluorescence anisotropy assay performed with synthetic peptides and with purified TIN2 confirmed that TRF2 phosphorylation at S410 increases the affinity between TRF2 and TIN2. When expressed in cells, the non-phosphorylatable TRF2-S410A mutant was able to interact with TIN2, which suggests that phosphorylation is not critically needed for mediating the interaction. On the other hand, the PLA assay revealed a stronger interaction between TRF2 and TIN2 upon inhibition of PPM1D that increases the level of TRF2 phosphorylation at S410. As TRF2 and TIN2 protect the ends of telomeres by promoting t-loop formation, we tested if the activity of PPM1D affects the architecture of the telomeric ends through regulating the shelterin complex assembly. To address this, we imaged the telomeres in psoralen-crosslinked chromatin spreads using Structured Illumination Microscopy and determined the fractions of linear and closed telomeres. Consistent with the published literature, we observed t-loops in about 25% of telomeres in parental cells . NeverthThe main finding of this study is that PPM1D is needed for DNA damage response at telomeric DSBs Figure . When PPPPM1D (We also noted that overexpression of PPM1D decreased the levels of TRF2 at telomeres which is in line with the disassembly of the shelterin after dephosphorylation of its components. However, we did not observe the formation of the TIFs upon overexpression of PPM1D, possibly due to the ability of PPM1D to efficiently suppress the activity of ATM ,80. We pPPM1D ,65,81,82gkac1269_Supplemental_FilesClick here for additional data file.


The resultingcrystalline solid catalyst—fully characterized with the helpof single-crystal X-ray diffraction—exhibits high catalyticactivity for the catalytic Buchner ring expansion reaction.The preparation of novel efficient catalysts—thatcouldbe applicable in industrially important chemical processes—hasattracted great interest. Small subnanometer metal clusters can exhibitoutstanding catalytic capabilities, and thus, research efforts havebeen devoted, recently, to synthesize novel catalysts bearing suchactive sites. Here, we report the gram-scale preparation of Ag 20 nanoclusters, which are efficiently stabilizedby the MOF channels. The small Ag20 nanoclusters, prepared in the gram scale,are used as efficient and recoverable catalysts for the Buchner ringexpansion reaction. These results expand the toolkit of readily affordableAg species for heterogeneous catalysis in organic synthesis.The present work describes the MOF-drivenpreparation ofwell-defined ligand-free Ag Moreover, the characterization of such tinySNMCs is really not an easy task and requires the use of high-resolutionmicroscopy techniques—for example, high-angle annular dark-field–scanningtransmission electron microscopy (HAADF–STEM)—whoseelectron beam can, with time, degrade the sample that is being observed.9 However, despite such complications, the outstandingmedical, optical, and/or catalytic properties of SNMCs make it wellworth the effort.11The preparation, stabilization, and characterizationof subnanometermetal clusters (SNMCs) has been a main challenge for chemists duringthe last years.12 In this context,a proper strategy to obtain ligand-free SNMCs consists in supportingthese metal species in porous solids such as zeolites or organic porousmaterials.13 More recently, another typeof porous materials, the so-called metal–organic frameworks(MOFs),14 have emerged as a suitable platformnot only to host SNMCs but also to be used as chemical reactors forthe in situ chemical synthesis of the SNMCs.16Focusing on the catalytic propertiesof SNMCs, stabilizing cappingligands of SNMCs dramatically reduces their catalytic activity asthey prevent a proper contact with reactants and trigger their decompositionunder reaction conditions. Therefore, it seems clear that “naked”SNMCs, with all metal atoms exposed, are called to offer the bestcatalytic performances.17 Moreover, aiming at encapsulating/synthesizingSNMCs, or even single-atom catalysts,22 MOFs offer clear advantages compared to other porousmaterials such as a fine control of the functionalities decoratingthe channels—which allows us to retain and align metals inspecific positions and controlled stoichiometries—and the possibilityto use single-crystal X-ray crystallography25 to unveil thecrystal structure of these ultrasmall metal species.MOFs are crystalline porous materials that have attractedsignificantattention in the past 2 decades due to the myriad applications theycan be used in.21 as a chemicalreactor, a highly robust anionic three-dimensionalMOF, with the formula NiII2{NiII4[CuII2(Me3mpba)2]3}·54H2O ,for the MOF-driven preparation of ligand-free tetranuclear [Pd4]2+ clusters after two consecutive post-syntheticsteps consisting of first replacing the Ni2+ cations hostedwithin its channels by Pd2+ ones and the concomitant reductionto form the final tetranuclear species within the empty space of theMOF. The resulting host–guest material had the following formula:[Pd4]0.5@Na3{NiII4[CuII2(Me3mpba)2]3}·56H2O 2]3}·54H2O (1) as the host matrix2]3}·51H2O (2) 2]3}·48H2O (3)  2b. Then,8H2O (3) 2c. The w3, containingAg2 clusters (together with unreduced Ag+ ions)has been further confirmed by the combination of a variety of techniquesincluding inductively coupled plasm–mass spectrometry (ICP–MS), elementalmapping, powder X-ray diffraction (PXRD), thermogravimetric analysis(TGA), X-ray photoelectron spectroscopy (XPS), and scanning electronmicroscopy (SEM). The N2 adsorption isotherms at 77 K confirmedthe permanent porosity of 2 and 3 (see below).Finally, as previously mentioned, SCXRD with synchrotron radiationallowed the resolution of the crystal structure of 2 and 3  and the observation of ultrasmall silver dinuclear entities andsurroundings within the solid in 3 (S1).The nature of the final hybrid material, lid in 3 2 and S1.II4CuII6 open-framework structures in both 2 and 3 are isoreticular and crystallize in the P4/mmm space group of the tetragonal system. Compound 2 exhibits the Ag+ cations situated within thewalls of the hydrophilic octagonal pores , where they are stabilized by noncovalent interactions involvingoxamate oxygen atoms [Ag+···Ooxamate of 2.72(1)–2.79(1) Å], with no evidence of previouslyNi2+ cations of 1, thus indicating that theyare completely exchanged by Ag+ ones or solvent water molecules [Ag+···O distancerange of 2.38(3)–2.56(3) Å], together with Ag+···Ag+ with a separation of 2.74(2) Å,which is shorter than the van der Waals contact distance, and theymight be considered as precursors of the Ag20 dimers observed in 3. Onthe other hand, the crystal structure of 3 reveals thestabilization provided by the nano-confined space of the MOF on theas-synthesized Ag20 dimers, constricted into the walls of the hydrophilic octagonalchannels of the anionic Ni42+Cu62+ open-framework net , togetherwith unreduced Ag+···Ag+ dimersin smaller square pores (S5). Further hydratedcharge-counterbalancing alkali Na+ cations are retainedin the preferential cationic sites, which stabilize the final material,showing an outstanding robustness . The poorer accessibility to the small square poresfor solvated NaBH4 is most likely the reason for stillunreduced Ag+···Ag+ dimers [blockedby Ag+···Ooxamate interactionsat a distance of 2.84(1) Å] . 20 dimers[intradimer Ag···Ag distance of 3.19(1) Å] arewell-fixed and stabilized inside the walls of the largest pores ofthe network by means of supramolecular interactions involving oxamateligands [Ag···Ooxamate distance range of2.93(1)–3.05(1) Å] and very weak connections with solventmolecules [Ag0···Owater distanceof 3.25(1) Å].The anionic NiAg+ ones 2b. Ag+ i84(1) Å] S5. Figur2 and 3 are given in Figures S6 and S7. EDX elemental mappings for Cu, Ni, Ag,and Na (3) elements show a heterogeneous spatial distributionof Ag atoms always located next to Cu and Ni atoms. Moreover, aberration-correctedHAADF–STEM (AC-HAADF–STEM) images are shown in Figure S8. They allow a direct visualizationof both Ag2 dimers together with Ag1 species—mostlikely to silver atoms residing in smaller square channels.SEM coupled with energy-dispersive X-ray spectroscopy(EDX) measurementsof 2 and 3  established the solvent contents for both materials, whichare reflected in their chemical formulas. PXRD patterns of 2 and 3  indicatethat the bulk samples are crystalline and pure, with no typical peaksof Ag0 nanoparticles. Indeed, experimental diffractionpatterns of 2 and 3 are identical to thetheoretical ones extracted from the SCXRD data. XPS spectra of compounds 2 and 3 are depicted in Figure S11. For 2, only possessing Ag+ cations,two bands at 367.6 and 373.6 eV, ascribed to Ag 3d5/2 andAg 3d3/2 binding energies, respectively,27 are observed .In turn, for MOF 3—where SCXRD and elemental analysessuggest that Ag+ cations and Ag20 nanoclusters coexist—apart fromthe same Ag 3d5/2 and Ag 3d3/2 bands at 367.6and 373.6 eV, respectively, indicative of Ag+, two additionalpeaks at 368.4 and 374.4 eV can be observed, which are attributedto reduced Ag0 atoms, with a 1:1 ratio respect to Ag+ . CO-probe diffusereflectance infrared Fourier transform spectroscopy (DRIFTS) was conductedon MOF 3, run at 77 K to avoid any in situ reductionof Ag+ and to observe potential Ag0–COspecies . The results showthree main peaks, one at 1938 cm–1, consistent withCO bridged-bonded to Ag0 atoms,28 a second at 2059 cm–1, attributable to Ag(CO)+ species,29 and a last peak at2043 cm–1, corresponding to free CO, after saturation.It is known that the adsorption of CO on Ag0 is lower thanAg+;30 thus, the lower intensityof the former makes sense and could very well correspond to a 1:1ratio between Ag oxidation states. These results strongly supportthat 50% of Ag+ present in 2 are reduced byNaBH4 forming Ag20 nanoclusters, whereas 50% of Ag+ cations remain untouched, occupying inaccessible sheltered interstitialpositions where the reducing agent cannot accede . The N2 and CO2 adsorption isothermsfor 1–3 confirm their permanent porosity. N2 adsorptionisotherms for 1–3, with calculatedBrunauer–Emmett–Teller31 surfaceareas of 974, 1013, and 625 m2/g, respectively, indicatea very similar permanent porosity for 1 and 3, which is in agreement with their identical estimated virtual diametersof 2.0 nm. In turn, MOF 2 exhibits lower N2 adsorption despite having the same virtual diameter (2.0 nm), whichcould be due to a partial collapse of the structure upon solvent evacuationtreatment. Remarkably, CO2 adsorption isotherms show a66% uptake increase for 3, suggesting quadrupole interactionsbetween CO2 molecules and Na+ cations.TGA of 3. The results show that the reaction betweentoluene (4) and ethyl diazoacetate (5) (EDA)proceeds rapidly (30 min) in a very high yield, under standard reactionconditions . Commercial Ag NPson alumina only gave a 16% conversion, and remarkably, the state-of-theart catalyst for this reaction, that is, Rh2(OAc)4, gave a lower result than MOF 3 in this study, underidentical reaction conditions (63% after the addition of 5 at once). An optimum >95% yield of product 6 wasobtainedafter maintaining a low concentration of 5 during thereaction, which was achieved by adding a solution of 5 (in dichloromethane) into the reaction mixture using a syringe pump,instead of adding it at once. Otherwise, the unwanted dimerizationreaction of 5 occurs. It is worth noting here that product 6 corresponds to the typical mixture of cycloheptatriene isomers,in accordance with previous results.32 A hot filtrationtest, where the solid MOF catalyst 3 is removed fromthe reaction mixture at the reaction temperature (60 °C) at anearly conversion (∼30%), shows that the catalytic active speciesare not present in solution within the experimental error . However,the catalytic yield of MOF 3 decreased to 30% after sixuses, which could be due to the progressive  leachingof active species during the reaction.The Buchner ring expansion reaction was attempted with catalyticamounts of nditions 4 top.21,eformer S15. Comm3 could be expanded to other aromatic substrates , cyano (product 15), methoxy (product 16), and ortho-disubstituted(product 18) aromatic compounds react with 5 in good to excellent yields and still in short reaction times (<2h). These results should be remarked upon as this is difficult tofind in the open literature Ag-catalyzed Buchner ring expansion reactions.35 The slightly lower results obtained for the bigger substrates 11 and 12 can be explained by the size discriminationassociated to the microporous structure of MOF 3. Tocheck this, 1,3,5-triisopropylbenzene 19 was tested asa substrate for the reaction, and product 20 was notfound . Besides, diffusiontests with varying stirring speeds confirmed that the initial reactionrate is dependent on the stirring, which confirms that the reactionoccurs inside the MOF’s channels . Indeed, 1,3,5-triisopropylbenzene 19 reactedwhen the Rh2(OAc)4 salt was used as a catalyst. Therefore,for MOF- or zeolite-stabilized Pd clusters,36 the formation of Ag2 clusters in micro-structured solidsenables not only the catalytic use of this metal in this complex organicsynthetic reaction but also its recovery and reuse.37 Interstitial Ag+ cations must be excluded as catalyticallyactive species since reagents cannot access these sites; besides,leaching does not occur under the reaction conditions employed. Finally,the integrity of MOF 3 is ensured by PXRD  and XPS  after catalytic experiments.The Buchner ring expansion reaction catalyzed by bstrates 1. Haloge20 clusters havebeen prepared, stabilized andcharacterized within an MOF and used as efficient and recoverablecatalysts for the Buchner ring expansion reaction. These results expandthe toolkit of readily affordable Ag species for heterogeneous catalysisin organic synthesis.In conclusion, ligand-free Ag2, which were suitable for XRD,were obtained by immersing crystals of 1 (ca. 0.0015mmol) for 48 h in 5 mL of a AgNO3 aqueous solution (0.004mmol), which was replaced three times. A multigram scale procedurewas also carried out by using the same synthetic procedure but withgreater amounts of both, a powder sample of compound 1  and AgNO3 , with the same successful results and a very high yield. Anal.: calcd (%) for Cu6Ni4Ag4C78H162N12O87 (3707.7):C, 25.27; H, 4.40; N, 4.53. Found: C, 25.34; H, 4.37; N, 4.59. IR(KBr): ν = 3008, 2961 and 2926 cm–1 (C–H),1601 cm–1 (C=O).Well-formeddeep green prisms of 2 (ca. 10 g) were suspendedin 50 mL of a H2O/CH3OH (1:2) solution, to whichan excess of NaBH4, divided in 26 fractions , was addedprogressively in the space of 72 h. After each addition, the mixturewas allowed to react for 1.5 h. After this period, samples were gentlywashed with a H2O/CH3OH solution and filteredon paper, giving high yields (ca. 98%). Anal. Calcd(%) for Cu6Ni4Ag4Na2C78H156N12O84 (3699.61): C,25.32; H, 4.25; N, 4.54. Found: C, 25.28; H, 4.17; N, 4.59. IR (KBr):ν = 3011, 2971 and 2928 cm–1 (C–H),1605 cm–1 (C=O).Both crystals (ca. 5 mg) and apowder polycrystalline sample of 2 and CO2 adsorption–desorption isotherms at 77 and 273 K were obtainedon polycrystalline samples of 2 and 3 usinga BELSORP-mini-X instrument. Samples were first activated with methanoland then evacuated at 348 K during 19 h under 10–6 Torr prior to their analysis.The N2 and 3 using a HITACHIS-4800 electron microscope coupled with an EDX detector. Data wereanalyzed using QUANTAX 400.SEM elemental analysis wascarried out for 3 was performed using an HAADF-FEI-TITAN G2 electronmicroscope. 5 mg of the material was re-dispersed in 1 mL of absoluteEtOH. Carbon-reinforced copper grids (200 mesh) were submerged intothe suspension 30 times and then allowed to dry on air for 24 h.HAADF–STEM characterizationfor 2 and 3 were introduced into 0.5 mm borosilicatecapillaries prior to being mounted and aligned on an Empyrean PANalyticalpowder diffractometer, using Cu Kα radiation (λ = 1.54056Å). For each sample, five repeated measurements were collectedat room temperature (2θ = 2–60°) and merged in asingle diffractogram.Polycrystalline samples of 2 and 3 were prepared by sticking, without sieving, the samplesonto a molybdenum plate using a scotch tape film, followed by air-drying.Measurements were performed on a K-Alpha XPS system using a monochromaticAl K source (1486.6 eV). As an internal reference for the peakpositions in the XPS spectra, the C 1s peak has been set at 284.8eV.Samples of 3. The experiments have been carried out in a homemade IR cell ableto work in the high and low (77 K) temperature ranges. Prior to COadsorption experiments, the sample was evacuated at 298 K under vacuum(10–6 mbar) for 1 h. CO adsorption experiments wereperformed at 77 K in the 0.2–20 mbar range. Spectra were recordedonce complete coverage of CO at the specified CO partial pressurewas achieved. Deconvolution of the IR spectra has been performed inthe Origin software using Gaussian curves where the full width athalf-maximum of the individual bands has been taken as a constant.The peak areas are normalized to the sample weight.DRIFTS using CO as a probe moleculewas used to evaluate the electronic properties of MOF 2 and 3 with ca. 0.06 ×0.08 × 0.08 and 0.08 × 0.12 × 0.12 mm dimensions, respectively,were selected and mounted on a MITIGEN holder in Paratone oil andvery quickly placed on a liquid nitrogen stream cooled at 90 K toavoid the possible degradation upon dehydration. Diffraction datafor 2–3 were collected using synchrotronat the I19 beamline of the DIAMOND at λ = 0.6889 Å. Crystallographicdetails can be found in the Supporting InformationCrystals of 3  was weighed in a 2 mL vial with a magneticstirrer, and the aromatic substrate (0.8 mL) was added. Then, thevial was placed in a pre-heated oil batch at 60 °C, and ethyldiazoacetate 5 (0.1 mmol) was added, either at once orusing a syringe pump (solution in dichloromethane). The mixture wasallowed to react for 0.5–2 h. After the reaction was complete,filtration was carried out to separate the solid catalyst. The reactionmixture was analyzed by gas chromatography (GC) and GC–MS.Further details can be found in the Supporting Information.MOF