Using the CFEL TapeDrive, minimization of sample consumption and data collection times needed for serial synchrotron crystallography (SSX) were achieved and the first room-temperature structures of two enzymes are described as well as an assessment of the benefit of SSX for a radiation-sensitive protein. Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance ( Other methods originally developed for SFX have been used in SSX experiments, such as viscous extrusion (LCP and other matrices)  and it is compatible with mix-and-diffuse methods for substrate or drug-design studies. Since it does not require modifications to the sample for delivery purposes, in the current setup, the sample vial  can be plugged directly into the liquid-dispensing system  in the presence of the long-lived intermediate-like 5-peroxy-9-methyl-isourate (5PMUA). Using this system, data collection times and sample consumption are similar to that of traditional single-crystal MX.The CFEL TapeDrive Fig. 1, developem Fig. 1 without 2.2.1.2.1.1.K. pneumoniae CTX-M-14 β-lactamase (CTX-M-14) was produced, purified and crystallized as described previously  CTX-M-14 solution (22 mg ml−1) was mixed with a 45%(v/v) crystallizing agent  and with a 5%(v/v) undiluted seed stock in batch crystallization setups. This resulted in crystals with a homogeneous size distribution of 11–15 µm after 90 min at 293 K. Crystals were centrifuged at 200g for 5 min and the supernatant was replaced with a stabilization buffer  to prevent further crystal growth. Prior to the measurements, the microcrystal suspension was filtered using a 30 µm gravity flow filter .2.1.2.N. haematococca GH11 xylanase (GH11) was produced and purified as described previously  were crushed under a stereomicroscope using a crystal crusher tool (Hampton research). The reservoir solution was pipetted to the drop and the seed stock was collected by washing the drop with reservoir solution. The seed stock was transferred to a seed bead tube , vortexed three times for 30 s each, with an interval of 30 s between each vortex to obtain the final seed stock. Protein solution (15 mg ml−1), crystallizing agent and seed stock were mixed with a ratio of 1:1:0.5. The mixture was vortexed 4 times for 30 s in 10 min intervals. After 30 min, the microcrystals were centrifuged at 200g and the supernatant was replaced with a precipitant solution. By applying the same protocol, microcrystals were obtained under two crystallizing conditions: crystallizing agent 1:1 M (NH4)2SO4, 100 mM sodium citrate pH 5.5, and crystallizing agent 2:200 mM (NH4)2SO4, 100 mM sodium citrate pH 5.5 and 20%(w/v) PEG 6000. Microcrystals obtained under both precipitant conditions – with dimensions between 10 and 20 µm – were tested for diffraction data collection.2.1.3.Aspergillus flavus urate oxidase (UOX) was expressed in E. coli using a codon-optimized synthetic cDNA  inserted into a pET24b vector. Protein expression was performed in E. coli BL21(DE3) cells at 20°C for ∼20 h. Protein purification was achieved using a combination of ammonium sulfate precipitation, DEAE and RESOURCE Q ion-exchange, phenyl sepharose hydrophobic interaction, and Superdex 75 size-exclusion chromatographic steps. For crystallization, UOX in Tris-acetate buffer  was concentrated to 20 mg ml−1 and saturated with 9-methyl uric acid (MUA). Large crystals were obtained at 20°C using the batch method by mixing the protein solution with an 8%(w/v) PEG 8000 reservoir in a 1:2 ratio under aerobic conditions. With this method UOX crystals typically reach dimensions of at least 400 × 400 × 300 µm. In the presence of O2 (air), MUA undergoes UOX-dependent dioxygenation, affording the mechanistically relevant 5PMUA peroxide derivative  and check_hkl , which are both part of CrystFEL.Data processing, starting directly from the native CBF-files, was carried out using the 2.4.6gth; Wiedorn et al., 2018phenix.refine . AceDRG derives atom types from the Crystallography Open Database  that play an important role in emerging multi-antibiotic resistance mechanisms . We had originally planned a longer data collection but X-ray loss due to a beam dump at PETRA III prevented further progression. Nevertheless, this ‘mini’ dataset afforded a total of 4286 indexed images from only ∼13.3 µl of sample injected. This 42.9% ‘indexing fraction’ of the total collected detector exposures corresponds to an effective data collection rate of about 10.7 Hz with only 32.2 ng of protein consumed per indexable detector frame. Processing with the s Table 1. Crystal1/2 cut-off of 0.15) extends to 1.4 Å (in comparison with 1.55 Å for the ‘mini’ data collection). This improvement in statistics does not translate in obvious changes in electron density maps . This corresponds to a 48.2% average ‘indexing fraction’ with 28.7 ng of protein consumed per indexable detector frame and a total volume of 91.4 µl microcrystalline suspension used. Data collection and refinement statistics are, as expected, superior for the combination of all runs, and the nominal resolution , albeit a bit larger than the RMSD between the structures from different numbers of indexed crystals from this study . The RMSDs between the structure from EuXFEL ]. Using the same parameters in phenix.refine and a high-resolution cut-off at 1.9 Å, Rfree values drop from 0.264 (1000 images) to 0.166 (40 000 images). The improvement in reasonable resolution and Rfree with the addition of more images merged, however, is not uniform, as can be seen in Fig. 4b). Rfree drops from 0.264 to 0.220, 0.209 and 0.189 when merging 4000, 5000 and 10 000, respectively, instead of 1000 detector images and only drops further to 0.166 for the full run (40 000 images). Similarly, the resolution at which CC* of the highest resolution shell reaches 0.5 drops from 1.83 to 1.65 Å using 4000 instead of 1000 images, and only drops to 1.58 Å at 10 000 images and 1.50 Å at 40 000 images [Fig. 4a)]. Similar progress can be observed when looking at overall values of CC* or Rsplit [Figs. 4c) and 4(d)]. Improvement in data quality can be best assessed when looking at the evolution of electron density maps with the addition of more indexed detector frames  for corresponding Rwork and Rfree] around Tyr41 are shown and clear improvement of map quality with the addition of more detector images can be observed. When looking at the 40 datasets, each consisting of 1000 detector images, within these datasets there is no clear trend, not with the number of indexable patterns per frame, the crystallographic figures of merit . The indexing rate is very high and ranges from 91.5 to 121.5% and Rfree values from 0.259 to 0.306, and the number of indexable frames and resulting Rfree values are not clearly correlated  goes from 1.83 Å down to 1.5 Å . In most cases the quality improvement was quite uniform across the 40 datasets, i.e. similar relative changes upon partiality refinement. Over all 40 datasets CC* increased by 0.029 ± 0.007 (from 0.910 ± 0.009 to 0.939 ± 0.008). However, there were some outliers, for example dataset 30, where overall SNR improved from 1.74 to 3.40, and overall CC1/2 changed from 0.71 to 0.81. Using automated refinement with the same conditions across all datasets for both conditions , the improvement through partiality refinement was less clear as indicated by the crystallographic metrics . In some cases, Rwork and Rfree did not improve for the same dataset after partiality modeling in CrystFEL; however, this was only true for 15% of cases . In one case Rfree remained the same and in 82.5% there were slight improvements through partiality modeling. The average Rfree over all 40 datasets dropped by 0.006 ± 0.001 (from 0.281 ± 0.002 to 0.275 ± 0.002) and the average Rwork dropped by 0.004 ± 0.001 (from 0.243 ± 0.002 to 0.239 ± 0.001).Partiality refinement in CrystFEL partialator with the The high and relatively uniform indexing rate and the absence of a trend in crystallographic or refinement statistics implies that, for these crystals at least, settling or other negative effects over the duration of one run cannot be observed and that TapeDrive can be used for stable and carefree data collection.B factor and the ADP from the refinement appear independent of the number of indexed crystals in a dataset. With the GH11 individual datasets, we could extend this investigation to the range from 1000 to 40 000 detector images. Using the same refinement strategy and input PDB, the average isotropic ADP drops slightly from 23.4 (1000 images) to 22.8 (40 000 images); however, again no clear trend is visible, as the ADP is 23.4 with 30 000 detector images .Additionally, we observed that, for the CTX-M-14 β-lactamase, the Wilson N. haematococca xylanase (GH11) the crystal symmetry does not change with crystal size. The crystals used for cryoMX  versus a = 79.45 Å, b = 38.50 Å, c = 53.59 Å, α = 90.00°, β = 91.43°, γ = 90.00° (cryo)]. The structural models resulting from refinement against the RT-SSX datasets are the first RT datasets of this N. haematococca xylanase. The structures superimpose very well with the structure from cryoMX  the addition of more images in an SSX dataset increases – as expected – the accuracy of the resulting model; but also (b) that the improvements, even with a 40 times longer data collection time, are rather minor. This shows that useful models, even those accurate enough for mechanistic studies, can be generated from the refinement of datasets of just 1000 detector images, corresponding to only 40 s data collection time.In the case of 3.3.3) using 1 mm-diameter glass beads to explore whether this rather coarse approach could be useful for SSX experiments, for example, when microcrystal optimization is problematic. We did not experiment with bead size but considered 1 mm beads a suitable choice as a previous report that used fragmentation for transmission electron microscopy analysis indicated that the use of 0.5 and 1.0 mm beads resulted in homogeneous populations of crystal fragments of low-micrometre sizes  (PDB entry 4cw2). We find that cell dimensions are modestly affected by cryo-cooling with a unit-cell volume contraction of about 2.1% . Overall, the present RT medium-resolution complex is virtually identical to that at 100 K and near-atomic resolution with an RMSD of 0.23 Å for all common main-chain atoms in the residue range (1–295) [Fig. 9a)]. The C-terminal region (296–301) is flexible and not visible in our RT structure although this is often the case even at cryo-conditions when the resolution is not as high as near-atomic.UOX crystallizes in space group et al., 2007b)] is very susceptible to radiolysis. At 100 K, an average diffraction-weighted dose (DWD) of about 200 kGy results in the complete rupture of the C5—O1 bond and values as low as 2.5 kGy are required to minimize this ]. As previously observed in the structure of the complex solved at cryo-temperature, 5PMUA binds at the interface between two protomers of the UOX tetrameric assembly in a cavity lined by K10*, I54*, A56*, T57*, D58*, F159, R176, L170, S226, V227, Q228, N254, H256, G286, I288 (the asterisk indicates residues belonging to a separate UOX chain) with the peroxide moiety sandwiched between the side chains of T57* and N254. Inspection of electron density maps obtained from the SSX experiment after refinement does not reveal obvious signs of 5PMUA radiolysis [Fig. 9c)]. The peroxide moiety displays pronounced pyramidalization at C5 accompanied by the strong distortion of its fused heterocycles. The C5—O1 bond length refines at the target value of 1.44 Å with no indication of C5—O1 rupture visible in mFo − DFc maps at the ±3σ level. We observe some negative density near the C5—O1 bond at the ±2.5σ level [Fig. S6(b)], however, this is matched by similar peaks in other regions of the maps thus suggesting that noise is a contributing factor at this threshold. Exact dose calculations are not straightforward for the present TapeDrive experiment because of complicating factors such as non-homogenous crystal size and variable crystal positioning with respect to the Gaussian-shaped beam. Nonetheless, DWD estimation using RADDOSE-3D , between 500 and 1000 datasets could, in principle, be collected within 12 h. Since one tape roll lasts for more than 12 h, one can collect these datasets without ever entering the hutch, and perform all experiments that are fully automated and even remote. Using an EIGER 16M at a frame rate of 133 Hz, instead of the PILATUS, up to 5400 datasets in 12 h are possible. Alternatively, if one wanted to extend the size of a dataset to 4000 detector images to more safely ensure a complete dataset, still more than 1000 datasets could be collected in 12 h, which is slightly more than the number of datasets possible at high-throughput cryoMX beamlines, and certainly more than what is currently possible for SSX with fixed targets. With this and further developments already under way, TapeDrive will enable autonomous and high-output data collection, from drug screening at physiological pH values and temperatures, to combined temperature-, concentration-, time- and pH-resolved 7D multidimensional investigations.10.1107/S2052252522010193/zf5019sup1.pdfFigs. S1-S6, RADDOSE-script and tables of data processing information. DOI: RT SSX structures:CTX-M-14, 7zpvPDB reference: CTX-M-14 (10k dataset), 8af7PDB reference: K. pneumoniae CTX-M-14 (5k dataset), 8af8PDB reference: N. haematococca GH11 xylanase (40k dataset), 8af4PDB reference: GH11 xylanase (10k dataset), 8af5PDB reference: GH11 xylanase (4k dataset), 8af6PDB reference: GH11 xylanase (1k dataset), 7zq0PDB reference: A. flavus urate oxidase complex, 7qarcofactor-free PDB reference:


CP1) in click reaction was evaluated to generate 1,4-disubstituted 1,2,3-triazoles selectively. The CP1 catalyst loading of 2 mol% was applied successfully in the reaction for primary azides with diverse functionalities of terminal alkynes in green solvent (EG/H2O). Moreover, the one-pot, multicomponent click reaction involving benzyl bromide, sodium azide, and phenylacetylene was also catalyzed by CP1. The findings show that 1D 2-Picolinic acid based Cu (II) coordination polymer catalytic systems are highly efficient for green click triazoles synthesis. DFT calculation supported the plausible mechanism involved in the CP1 catalyzed click reaction.The catalytic activity of 1D 2-Picolinic acid based Cu (II) coordination polymer ( Many 1,2,3-triazole compounds are clinically important and exhibit various biologically important activities such as antibiotic4, anti- Human Immunodeficiency Virus drug5, anticancer6, antiviral7, etc. As a result, there is immense interest in the emerging synthetic protocols for these heterocycles8. Huisgen 1,3-dipolar azide/alkyne cycloaddition is the conventional method for 1,2,3-triazoles synthesis. However, the classical thermal conditions which does not use a catalyst requires high temperature and long reaction time, which results in yielding a mixture of 1,4 and 1,5-substituted triazoles without regioselectivity9. The Cu-catalyzed Huisgen 1,3-dipolar azide/alkyne cycloaddition, popularly known as ‘Click reaction’, which was found independently by Sharpless10 and Meldal11 groups, is considered to be the most powerful approach for 1,2,3-Triazoles preparation. This transformation speeds up and enables the milder reaction conditions with a wide substrate scope and absolute regioselectivity . Numerous researches have suggested that copper source which can create the catalytically active Cu(I) species in the reaction media can possibly initiate the click reaction. In general, the commonly used catalytic system mostly make an extensive use of Cu(II) salt and reducing agent (e.g. sodium ascorbate12 or hydrazine hydrate13) in water or mixture of water with an organic solvent. However, the difficulty in removing the copper ions from the synthesized products using the commonly practiced catalytic systems limits its application in pharmaceutical and biological sciences14. To overcome this problem, many new alternative catalytic materials have been developed by various researchers over the past years. An iron based Cu nanoparticles catalytic system for click reaction was developed by Lipshutz and coworkers15. A nano Cu-catalyst in polydiacetylene micelles was reported by Doris et al.16 for Huisgen cycloaddition reaction. Recently, a nanofibrous copper (0) catalytic system for azide/alkyne cycloaddition reaction was reported by Sayyahi et al.17. In 2019, Esmaeilpour et al.18 have introduced a reusable Fe3O4@SiO2-dendrimer-encapsulated Cu(II) catalyst for CuAAC reaction. In spite of these considerable achievements from various research studies in the recent past, most of the present systems of catalysis have limitations for practical applications because of the requirement for expensive additive, organic solvent, high catalyst loading and long reaction time. Therefore, it has become indispensable to develop new systems of CuAAC reaction which is efficient, economical, greener, and low Cu-loading.1,2,3-Triazoles are a vital category of five-membered heterocycles that are increasingly found in diverse applications in chemistry, biology, medicine, and materials science19. Gas storage21, drug delivery23, photocatalysis25, magnetism27, luminescence29, and catalysis31 are some of the uses of these hybrid materials. Numerous catalytic studies on the potential uses of 2D and 3D CPs have been reported in reactions like Knoevenagel reaction33, oxidation35, photocatalysis36, Heck reaction37, and Click reaction38. However, the potential applications of one-dimensional CPs were given lesser attention. Despite their structural simplicity, the 1D CPs are comparatively much easier to synthesize and their topological structures can be tuned more effectively to maximize the scope in different applications40. Thus, more effort should be made to systematically study the catalytic application of 1D CPs. The carboxylate ligands are usually employed in designing the CPs because of their diverse coordination modes and bridging ability. Copper(II) carboxylates, especially those with nitrogen donor ligands, have been extensively investigated because of their intriguing topological structures and properties.In the recent decades, coordination polymers (CPs) that are formed from organic bridging ligands with transition metal ions have evolved as one of the fastest emerging area of chemical studies because of their remarkable structural features and distinctive functionalities41 the structural characteristics and photocatalytic activity of 1D 2-picolinic acid based Cu (II) coordination polymer formulated as {[Cu(2-pic)2]2H2O}n (CP1) (2-pic = pyridine-2-carboxylic acid) has been reported. To the best of our knowledge CP1 has not been explored for catalytic application in organic synthesis. Intrigued by its interesting structures, and properties and also considering the need to develop new catalytic system for CuAAC reaction with low Cu-loading, economical, and greener, we took interest in investigating the catalytic property of CP1 in the synthesis of 1,2,3-triazoles. This study presents a sustainable chemical protocol for the “click reaction” by employing CP1 as the catalyst that can selectively provides 1,4-substituted triazole, instead of 1,5-substituted triazole. The reaction mechanism was further elucidated by DFT study. The catalytic property of CP1 system under the influence of various parameters has also been investigated. The successful synthesis of one-pot multicomponent cycloaddition of benzyl bromide, phenylacetylene, and sodium azide adds further advantage to this catalytic system.In our recent publication254 plates, which were then visualized under UV light (λ = 254 nm). Column chromatography method was used for purification of the products using silica gel (60–120 mesh). FTIR spectra of all the isolated 1,2,3-Triazole products were recorded on Agilent Cary 630 FTIR spectrometer. 1H NMR (500 MHz), and 13C NMR (125 MHz) were scanned on Bruker Avance III 500 MHz FT-NMR spectrometer in CDCl3 taking TMS as internal standard. Chemical shifts (δ) are shown in ppm, and the values of coupling constants (J) are expressed in hertz. Splitting patterns are denoted by the following abbreviations: s/singlet, d /doublet, t/triplet, q/quartet, and m/multiplet.All the chemicals used in this study were procured from Sigma-Aldrich/TCI, Japan and organic solvents were distilled in accordance with the standard procedure as required. The reactions performed in the present study were carried in oven-dried round-bottom flask in open air. Thin-layer chromatography was used to monitor progress of all the reactions by utilizing silica-gel 60FCP1 was prepared following the same procedure as reported in our previous work41. A solution of Picolinic acid  in 5 ml of 1NHCl was added to a stirred solution of CuSO4.5H2O  in 5 ml of 1NHCl. After stirring for 30 min at 70 °C the blue solution was let to cool in ice water which resulted in the formation of dark blue crystals. The crystals were filtered and then recrystallized with ethanol, and finally dried at 60 °C in oven for 40 min to produce 40% Yield. Anal. Calc. for [C12H8N2O4Cu]0.2H2O: C, 41.92; H, 3.49; N, 8.15. Found: C, 41.25; H, 3.02; N, 8.18.The CP1 (2 mol%), and 2 ml EG/H2O (1:1), and was stirred at 30 °C for 4 h. TLC was used in monitoring the reaction’s progress, and upon completion the mixture was extracted with (4 × 10 ml) ethyl acetate. Na2SO4 anhydrous was used for drying the organic layer, which was then concentrated under reduced pressure, and subsequently purified using column chromatography (n-hexane/ethyl acetate) to provide the targeted product 3a-3n (for details see SI).Mixture for the reaction was prepared in a 50 ml round-bottom flask using azide (1.0 mmol), alkyne (1.2 mmol), sodium ascorbate (3 mol%), H-1,2,3-triazole (3a): White solid; Yield; IR (νmax cm−1): 3126, 3059, 2923, 1607, 1464 (CH2), 1440, 1425, 1355, 1222 (N–N = N-), 1194 (C-N), 1073, 1047, 974, 912, 825 , 763, 725, 691, 578. 1H NMR : δ = 7.81 , 7.66 , 7.42–7.36 , 7.34–7.29 , 5.58 . 13C NMR : δ = 148.33, 134.75, 130.60, 129.26, 128.89, 128.26, 125.78, 119.55, 54.33. HRMS (ESI) calcd for C15H13N3 [M + H]+: 236.1109, found: 236.1186.1-Benzyl-4-phenyl-1CP1 (2 mol%), and 4 ml of EG/H2O (1:1) were mixed in a 50 ml RB flask, and was stirred at 30 °C for 4 h. TLC was used in monitoring the reaction’s progress, and upon completion the mixture was extracted 4 times with ethyl acetate. Na2SO4 anhydrous was used to dry the organic layer, which further was concentrated under reduced pressure, and subsequently purified using column chromatography (n-hexane/ethyl acetate) to provide the 1-benzyl-4-phenyl-1H-1,2,3-triazole.Benzyl bromide (1 mmol), sodium azide (1.1 mmol), phenylacetylene ( 1.2 mmol), 3 mol% of sodium ascorbate (3 mol%), 2 mol% of CP1) were generated using Bruker Nonius SMART APEX II diffractometer equipped with a Charge-Coupled Device (CCD) area detector and graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) operating at 296 K. Crystal structure refinement was performed with SHELXL-14/7 program42 using the full-matrix least-squares method on F2. All the non-hydrogen atoms of CP1 were refined anisotropically against F2 of all reflections. The H-atoms of the organic ligands were placed at their geometric positions and refined isotropically. CCDC 2,094,267 contains the crystallographic data for CP1.The SCXRD data of 1D Copper-based coordination polymer / transition states (one imaginary frequency) at the identical theoretical level. The effects of solvation or dispersion forces were not taken into account in our calculations because they probably differ from those observed in real compounds. Literature on the theoretical features of CuAAC reaction mechanism suggested that these effects do not seem to be the critical parameters/characteristics for those general reactions that work experimentally for a family of reactive compounds. The Gibbs free energy at 298.15 k was calculated using frequency calculations. The TS was optimized applying Berny algorithm (opt = ts) at the identical theoretical level.The geometry optimization for all the interested compounds, including the intermediates and transition states, was carried out in the gas phase using the DFT computations with B3LYP/631GCP1 (2 mol%) as homogeneous catalyst, Na ascorbate (3 mol%) in water under open-air condition at 30 °C, which led to 60% yield of 1-benzyl-4-phenyl-1H-1,2,3-triazole after 4 h (Table CP1 (2 mol%) and Na ascorbate (3 mol%) in the CuAAC cycloaddition reaction. The reaction proceeded in aprotic solvents like DMF, CH3CN, DMSO, and THF, but resulted in inferior yields (entries 2–5). On the contrary, the reaction proceeded well with protic solvent MeOH and resulted in a 53% yield of the product 3a (entry 6). It’s worth noting that when the reaction was tested in ethylene glycol, the yield increased significantly (entry 7). On further evaluation it was found that mixed solvents of EG/H2O gave the best activity with considerable increase in the yield (entry 10). With EG/H2O as the optimal solvent we proceeded to examine catalyst loading for the reaction and discovered that lowering the catalyst loading to 0.5% leads to a decline in the conversion . It was also found that there was no significant improvement in the yield of the corresponding 1,4-disubstituted 1,2,3-triazole by increasing the catalyst loading to 3%, . The reaction failed in the absence of CP1 (entry 15). We also investigated the reaction in absence of Na ascorbate and the product conversion was found to be in trace amount only (entry 16). Therefore, the reaction conditions in entry 10 have been evaluated to be the most suitable one for establishing a method which has broader application with lower reaction times. These findings suggested that strong reactivities in these catalytic systems required both CP1 and Na ascorbate.We initiated our investigation of 1D Copper (II) coordination polymer-catalyzed cycloaddition using benzyl azide and phenyl acetylene as the model substrates. The reaction was tested in presence of 4.5H2O and Cu(II) complex were used as catalyst (entries 17–18). This result suggests the significance of CP1 in promoting CuAAC reactions.We have also screened other copper catalysts under the same reaction conditions, and the optimization studies have shown that moderate yields of the product were formed when CuSO1a with phenylacetylenes consisting of electron-donating substituents  proceeded effectively to afford the corresponding 1,4-disubstituted-1,2,3-triazole in 61–88% isolated yields  aromatic alkynes on reaction with 1a shows a negative influence providing 50–64% isolated yields of the 1,4-disubstituted-1,2,3-triazole products  cycloaddition reactions. We also examined our catalytic system for the one-pot, multi-component CuAAC reaction involving in situ formation of organic azide from aryl bromide with NaN3. Benzyl bromide was used as the precursor since the benzyl azide utilized in this study was prepared from the corresponding bromide. The CP1 catalyst worked effectively for the multicomponent model reaction of benzyl bromide, NaN3, and phenylacetylene at the optimized reaction conditions, and generated the desired triazole product with high yield , benzyl azide (4.0 mmol) and Na ascorbate (3 mol%) in the presence of CP1 (2 mol%) in EG/H2O (16 ml) for 4 h. After the completion of the reaction, ethyl acetate was added to the reaction mixture to extract the product. The organic layer having the product and the EG/H2O layer having the homogeneous catalyst CP1 were then separated. For the reusability study, the isolated EG/H2O layer was reemployed with addition of Molar quantities of reactants, and the experiment was allowed to proceed under the same reaction conditions. The first recycle reaction yielded 84% product  with sodium ascorbate, the reaction can be activated by coordinating the alkyne with the cationic Cu(I) center in a η2 mode. The –CH group in the alkyne moiety gets deprotonated by one of the copper coordinated picolinic acid ligands, resulting in the creation of a Cu(I)–acetylide complex. The organic azide further coordinates with the cationic Cu(I)–acetylide complex and transforms into a six-membered ring metallo-cycle, followed by contraction of the six-membered ring to produce a copper–triazolide  centre by displacing one of the picolinic acid ligand resulting in a η2-alkyne complex. This Cu-acetylide complex was used as an initial reactant in Fig. 2) by coordinating to the metal centre through the methylated nitrogen atom. The reaction of methyl azide with η2-alkyne complex to generate 2 was calculated to be slightly exothermic by 0.2 kcal/mol. Thereafter, the azide’s distal nitrogen in 2 binds the acetylide’s C-2 carbon creating the six-membered metallocycle 3. This phase is endothermic and the computed barrier is 14.4 kcal/mol, which is also much lower than the reported mononuclear copper acetylide barrier 53. The ring contraction barrier, from 3, which forms the copper-triazolide complex 4 via TS3/4 is 14.1 kcal/mol. TS3/4 is 0.3 kcal/mol lower than TS2/3. The final step involves a rapid protonation of the copper-triazolide, which results in the triazole product; in the meantime, CP1 active catalyst regenerates to complete the catalytic cycle. The geometries of the optimized intermediates, TS2/3, and TS3/4 are given in Fig. We have theoretically evaluated our hypothetical mechanism based on the proposed mechanism using the DFT calculations on a simplified system constituted of CP1 catalyst loading of 2 mol%. The one-pot, multicomponent cycloaddition involving benzyl bromide, sodium azide, and phenylacetylene was also efficiently catalyzed by CP1. At low temperature (30 °C) the CP1 catalytic system was adequately applied in selective construction of 1,4-disubstituted triazoles. The results suggest that CP1, which is simple to prepare, reusable and has a high substrate tolerance, can make considerable contribution to an extensive range of applications. Moreover, the DFT calculations were performed to support the experimental results of the proposed mechanism.In conclusion, we have employed a 1D Pyridine-2-carboxylic acid based Cu (II) coordination polymer as the active catalyst in green click triazoles synthesis with Supplementary Information.


Enterococcus faecium has become an important drug-resistant nosocomial pathogen because of widespread antibiotic abuse. We developed short and chemically simple antimicrobial peptides (AMPs) with a selective amino acid composition, fixed charge, and hydrophobicity ratio based on the core antimicrobial motif of bovine lactoferrin (LfcinB6). Among these peptides, 5L and 6L (both 12 residues long) demonstrated a narrow spectrum and high antibacterial activity against drug-resistant E. faecium isolates with a minimal inhibitory concentration (MIC) that ranged from 4–16 µg/mL. At 32 µg/mL, peptides 5L and 6L inhibited E. faecium strain C68 biofilm formation by 90% and disrupted established biofilms by 75%. At 40 µg/mL, 5L reduced 1 × 107E. faecium persister cells by 3 logs within 120 min of exposure, whereas 6L eliminated all persister cells within 60 min. At 0.5× MIC, 5L and 6L significantly downregulated the expression of a crucial biofilm gene ace by 8 folds (p = 0.02) and 4 folds (p = 0.01), respectively. At 32 µg/mL, peptides 5L and 6L both depolarized the E. faecium membrane, increased fluidity, and eventually ruptured the membrane. Physiologically, 5L (at 8 µg/mL) altered the tricarboxylic acid cycle, glutathione, and purine metabolism. Interestingly, in an ex vivo model of porcine skin infection, compared to no treatment, 5L (at 10× MIC) effectively eliminated all 1 × 106 exponential (p = 0.0045) and persister E. faecium cells (p = 0.0002). In conclusion, the study outlines a roadmap for developing narrow-spectrum selective AMPs and presents peptide 5L as a potential therapeutic candidate to be explored against E. faecium. Enterococcus faecium is the most common cause of central line-associated bloodstream infections with 70% being VRE  dye . We calculated the percentage of biofilm growth by assuming that there was 100% biofilm growth in wells with bacteria without peptide treatment.We evaluated the ability of the lactoferrin-derived peptides to inhibit biofilm formation following an established protocol with modifications . In shorE. faecium strain C68 (adjusted to 1 × 108 CFU/mL) in BHI media (supplemented with 0.2% glucose) from overnight grown cultures. We added 100 µL of the bacterial culture to wells of flat-bottomed 96-well polystyrene microtiter plates . We then incubated the plates at 37 °C for 24 h in static conditions to allow biofilm formation. After incubation, we carefully pipetted out the media and washed the wells with PBS  to remove loosely attached planktonic cells. We treated the established biofilms in each well with 10 µL of serially diluted 10× peptide solution, followed by the addition of 90 μL of BHI media (supplemented with 0.2% glucose). The plates were incubated for another 24 h at 37 °C before processing for biomass and live-cell contents. We quantitated the concentration of peptide that disrupts 50% of established biofilms (MBEC50).We prepared exponential cultures of E. faecium strain C68 (adjusted to 1 × 106 CFU/mL) in BHI media (supplemented with 0.2% glucose) from overnight grown cultures. To establish biofilms, we added 1 mL of the bacterial culture to the chambers of cuvettes  and maintained them at 37 °C for 24 h under static conditions [We prepared exponential cultures of nditions . We careE. faecium strain C68 adjusted to an OD600 = 0.4 in BHI media from overnight grown cultures. After treating 10 mL of bacterial culture with 0.5× MICs of 5L and 6L for 30 min, we extracted RNA using the RNeasy mini kit  according to the manufacturer’s instructions. Using the primers listed in For the qPCR of genes associated with biofilm formation, we followed an established protocol for RNA isolation and quantitative PCR reactions with minor modifications . We prepE. faecium strain C68 culture was grown to the stationary phase and then treated with gentamicin at 400 μg/mL for 5 h. We then disposed of the antibiotic-containing media and regrew the bacteria for another 18 h in fresh media. To produce stable persister cells, we repeated the cycle of antibiotic exposure and regrowth five times. The bacteria culture was then washed with the same volume of phosphate-buffered saline (PBS) 3× times and adjusted to 1 × 107 CFU/mL in PBS. For killing kinetics of the persister cells, we added 1 mL of the cell suspension to the wells of a 2 mL deep well assay block  containing 10× MICs of 5L and 6L and incubated them at 37 °C, with shaking at 225 rpm. At specific times , we collected 20 μL samples, diluted them serially, and then plated them on tryptic soy agar (TSA)  plates for 18 h at 37 °C for colony counting. We performed the experiments in duplicate. As a comparison, we prepared the exponential cells of E. faecium strain C68 in PBS and analyzed their killing kinetics in the same manner as described for persister cells. For the generation of antibiotic-induced persister cells, we followed an established protocol . In brieE. faecium strain C68 persister cells (described in the above section) and diluted them in PBS to OD600 = 0.2. We added SYTOX Green  to the diluted persister suspension to a final concentration of 5 μM and incubated them for 30 min at room temperature in the dark. We added 90 µL of the dye/bacteria mixture to 10 μL of serially diluted 10× peptide concentration in a 96-well black, clear-bottom plate  and recorded fluorescence on a SpectraMax i3x  with excitation and emission wavelengths of 485 nm and 525 nm, respectively, for 1 h at room temperature. For comparison, we also conducted membrane permeability assays using exponential cells and followed similar protocol as described for persister cells.We generated E. faecium strain C68 in fresh BHI media, washed it twice in PBS, and resuspended it in a double volume of PBS. To energize the bacterial cells, we added 25 mM of glucose for 15 min at 37 °C, followed by 500 nM of DiBAC4(3) trimethine oxonol) . The DiBAC4(3) is a potential sensitive probe that enters depolarized cells, binds to intracellular proteins or membranes, and exhibits enhanced fluorescence. We distributed 90 µL of this bacterial culture in a 96-well black plate with a clear bottom  and monitored the fluorescence for 20 min at excitation 485 nm and emission 520 nm, respectively, until the baseline was stable. Then, we added 10 µL of serially diluted peptide solutions and recorded the fluorescence for another 40 min. We included Triton X-100 (1%) as a positive control. We measured bacterial membrane potential based on an established protocol with minor modifications . We prepE. faecium strain C68 bacteria three times with PBS and resuspended in half the volume of the initial culture taken for washing. To the bacterial suspensions in PBS, we added the Laurdan dye  at a final concentration of 10 µM at room temperature in the dark and distributed 100 µL of this dye/bacteria mixture into 96-well transparent black plates  containing a 10× peptide solution. After incubation at room temperature for one hour in the dark, we measured the fluorescence intensity using a SpectraMax i3x , with excitation at 350 nm and dual emissions at 435 nm and 490 nm. Accordingly, we calculated the Laurdan generalized polarization (GP) using the formula GP = (I435 − I490)/(I435 + I490). Our positive control included 50 mM of benzyl alcohol as a membrane fluidizer.We measured the peptide-induced changes to the bacterial membrane fluidity following an established protocol . BrieflyE. faecium strain C68 bacteria three times with PBS and adjusted it to OD600 = 0.4 in PBS. We added PI dye to the bacteria to achieve a 2 µM final dye concentration and distributed 90 μL of this bacteria/dye mixture into a 96-well black clear-bottom plate  containing 10 μL of serially diluted 10× peptide concentrations. Subsequently, we monitored the plate fluorescence on a SpectraMax i3x  with an excitation wavelength of 584 nm and an emission wavelength of 620 nm for 1 h at room temperature.We used a fluorescence-based bacterial permeation assay to establish membrane-oriented interactions of the designed peptides. Briefly, we washed the exponential phase of E. faecium strain C68 in BHI media. We inoculated the bacteria in a fresh BHI medium the next day to achieve the exponential phase and adjusted the bacterial density to 1 × 108 CFU/mL. We added 90 µL of this bacterial culture to each well of the microtiter plates  containing 10 µL of serially diluted 10× peptide solution. We monitored the bacterial growth on a SpectraMax i3x spectrophotometer  by taking OD600 readings every 10 min for 4 h. We cultivated overnight cultures of E. faecium strain C68 bacteria three times with PBS (pH 7.4) and adjusted it to OD600 = 0.4 in PBS. Following treatment of the bacteria with 32 µg/mL of 5L and 6L peptides at 37 °C for 30 min, we centrifuged the cells at 14,000 rpm for 5 min. We transferred 50 µL of the supernatant of each sample to each well of black, clear-bottom, 96-well plates  containing 50 µL of BacTiter-Glo reagent  and measured plate luminescence after 5 min of incubation at room temperature in the dark with a SpectraMax i3x reader .To determine the ATP leakage potential of the peptides, we employed a luciferase-based assay detailed in . In brieE. faecium that was determined previously using biochemical lipid extraction methods [We performed molecular dynamics (MD) simulations in the presence of POPE: cardiolipin (3:1) membranes with 128 molecules of the desired lipid system. We used the lipid composition of  methods . We cons methods . We plac methods . We init methods ,37 and fE. faecium strain C68 grown in BHI media adjusted to OD600 = 0.5. We treated 10 mL of this bacterial culture with 2× MIC of peptide 5L or water  for 30 min at 37 °C . After that, we washed the bacteria 3× times with PBS and stored the pellet at −20 °C. At a later stage, to each cell pellet, we added 0.2 mL of 2-propanol: 100 mM of NH4HCO3, pH 7.4 (1:1 v/v), and sonicated the cells. For spiking, we added 20 µL of IS to each sample and vortexed them. Subsequently, we added 1.0 mL of methanol for deproteinization and cooled the mixture at −20 °C for 10 min. After centrifugation for 10 min at 14,000× g at 4 °C, we transferred the supernatants into glass tubes and dried them under a stream of nitrogen at 40 °C. Finally, we dissolved the residues in 100 µL of water and injected 3 µL of this solution into an LC-MS/MS 8060 system , equipped with a DUIS source operated in both positive and negative electrospray ionization modes. For the liquid chromatographic analysis, we used the Nexera UPLC system . We quantitated primary metabolites (~150) following an established LC-MS/MS method [For the metabolomics sample preparation, we used exponential cultures of S method .g for 5 min, transferred 60 µL of the supernatant into a fresh 96-well plate, and read the absorbance at 540 nm. We calculated the percent hemolysis considering 100% hemolysis caused by 1% Triton X-100, and 0% on PBS and using the formula for percentage of hemolysis as: (A540 nm in the peptide solution − A540 nm in PBS)/(A540 nm of 1% Triton X-100-treated sample − A540 nm in PBS) × 100.We evaluated the ability of the peptide to cause hemoglobin leakage using the method described in . In brie2 in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin . Next, we harvested and resuspended the cells in a fresh medium and distributed 50 µL (1 × 106 cells) in a 96-well plate containing 50 µL of serially diluted AMPs in serum and antibiotic-free DMEM and incubated the plates at 37 °C in 5% CO2 for 24 h. Before the end of the incubation period (at the 20th hour), we added 10 µL of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium (WST-1)  to each well and monitored the reduction of WST-1 at 450 nm using a SpectraMax i3x . We performed the assays in triplicates and calculated the percentage of cell survival.We used liver-derived HepG2 cells to evaluate AMP cytotoxicity behavior in mammalian cells based on an established protocol . We main6 CFU of exponential or persister E. faecium strain C68 cells in a total volume of 10 µL. Following an incubation of 1 h at 37 °C, we treated the skin with 50 µL with 5L and 6L at concentrations of 10× or 1× MIC. After an additional 4 h of incubation at 37 °C, we washed each tubing well with 250 µL of PBS (pH 7.4) containing 0.05% Triton X-100 to remove bacteria. After serially diluting the bacterial sample, we counted their CFUs.We evaluated the efficacy of designed lactoferrin AMPs in an ex vivo porcine skin model previously described with minor modifications . We first-test. For all of the experiments, we considered p < 0.05 as significant.We used one way ANOVA for the statistical analysis in all of the experiments except for real-time PCR and metabolomics where we employed Student’s E. faecium. Therefore, we aimed to improve the hydrophobicity of the LfcinB6 peptides.To design simple and short AMPs for further antibacterial agent development, we considered peptides with only one or two copies of the LfcinB6 core antimicrobial motif. The native LfcinB6 peptide has a net charge of +3 and a hydrophobic content of 33%. Despite the three Arg residues that can bind effectively to bacterial membranes, the peptide is inactive against E. faecium strain C68. We used a single copy of LfcinB6 to design AMPs with increased hydrophobic contents of ~60% . To incrE. faecium strain C68 with MIC values of 4 µg/mL. The designed lactoferrin peptides did not show antibacterial activity toward S. aureus and E. faecalis, except peptide 6L with a very high MIC of 32 µg/mL against S. aureus MW2 (The short peptide 2L (seven amino acid residues) was ineffective (tested up to 32 µg/mL), so we decided to use two copies of LfcinB6 for the next round of peptide design. Two copies of LfcinB6 have 12 amino acid residues imparting 33% hydrophobicity, and in order to maintain 58% hydrophobicity, three hydrophobic substitutions are required. As a result, we replaced both Gln4/10 with Leu, followed by a sequential Arg to Leu substitutions  in successive peptides. Peptide 3L with arg1 replaced with Leu had an MIC of 16 µg/mL. Other AMPs (4L–8L) with Arg to Leu substitution showed excellent antimicrobial activity against reus MW2 . 4, and 1 mM of MgSO4, the MIC of peptides 1L–8L (except 2L) was influenced by only 2-fold  while the MICs of peptides 5L–8L were only increased by 2–4 folds. Next, to evaluate the robustness of the antibacterial activity of the designed LfcinB6 peptides, we included physiological salts into the MIC assays. In the presence of 150 mM of NaCl, 8 mM of ZnSOy 2-fold . HoweverE. faecium ranged from 4–16 µg/mL, while that of peptide 8L ranged from 8–32 µg/mL against vancomycin-sensitive strains D14, D24, D25, and D29 and vancomycin-resistant E. faecium strains C68 and WC176 (VRE) (E. faecium strain (E007).To further evaluate the antimicrobial potency of the LfcinB6 peptides, we tested the activity of peptides 5L–8L against drug-resistant clinical isolates and other bacterial physiological forms, such as biofilms and antibiotic-induced persister cells. The MICs of peptides 5L–7L against clinical isolates of 76 (VRE) . HoweverE. faecium strain C68 (50 1 µg/mL), followed by 8L (MBIC50 2 µg/mL) reduced the biomass contents , 6L depleted 92% of the biomass, followed by 5L (90%), 7L (85%), and 8L (80%). In the more robust condition of 24 h established biofilms, the peptide 5L killed 50% of the live bacteria contained within the matured biofilm matrix C. Peptid16 µg/mL D. PeptidE. faecium E007 (a tetracycline-resistant strain) (50 10 µg/mL) and 6L (MBEC50 8 µg/mL) disrupted 75% of the established biomass, while peptides 7L and 8L disrupted only 25–30%. Additionally, to verify the robust antibiofilm properties of peptides 5L and 6L, we examined their ability to disrupt 24 h established biofilms of  strain) . Indeed,E. faecium strain C68 biofilms  killed most of the bacteria inside the biofilms. Untreated biofilms E were grbiofilms F,G are dE. faecium strain C68. The peptides 5L and 6L at subinhibitory concentrations (0.5× MIC) suppressed the expression of crucial biofilm genes (ace (8-fold), esp (4-fold), ebpA (2-fold), and ebpC (2-fold). Whereas 6L significantly downregulated the expression of ace (4-fold) and ebpC (2-fold).In addition, to uncover the mode of antibiofilm activities of peptide 5L and 6L at the genetic level, we measured the relative expression of significant biofilm-regulating genes in lm genes H. Peptid7 CFU of a culture with E. faecium strain C68 persister cells by 3 logs, while peptide 6L was even more active  dye (described in Methods). E. faecium strain C68 cell membrane exposure to peptides 5L–8L (at 16 µg/mL) induced a rapid fluorescence rise, indicating change in transmembrane potential . In the principal component analysis (PCA) and partial least squares-discriminant analysis (PLSDA) plots, we found distinct metabolic alterations between the E. faecium control and 5L-treated conditions with three technical replicates  agrees with our previous observations on cecropin-4-derived peptides, where we found that a minimum amino acid length is required in AMPs to maintain their antimicrobial activity [A previous physicochemical analysis of 138 AMPs active against of 58.2% . Thus, iactivity . Moreoveactivity  and APO-activity , each wiE. faecium. Salt-resistant linear peptides such as LfcinB6-KR-12 hybrid peptide, cecropin-4-derived C18, and arginine-rich decamer peptides D5 and D6 have high hydrophobicity and charges like LfcinB6-designed peptides, suggesting salt sensitivity in AMPs is template-dependent [+ and divalent Zn2+, Mg2+, and Ca2+ cations.Salt decreases the potency of AMPs due to charge screening effects as both AMPs and salts compete for bacterial membrane binding . For exaependent ,18,49. HE. faecium. Notably, peptides 5L and 6L did not only kill persister cells of E. faecium but also inhibited the biofilm formation process and disrupted 24 h established biofilms  model within 25 ns. Interestingly, a membrane-active lipopeptide of lactoferrin, acyl-LfcinB6, also binds to 3:1 POPE: POPG membrane within 75 ns, thus, confirming the membrane-targeting mechanism of LfcinB6 peptides [LfcinB6 is amphipathic with Arg residues present on the hydrophilic face, and Trp residues making up the hydrophobic face . The Lfcsruption . Cationiconstant . In agrepeptides . E. faecium  [In addition to targeting the bacterial membrane, LfcinB peptides also affect ribosomes, mitochondria, and other physiological functions ,67. We p faecium . Antibacxidation . Additiorgeting) . Furtherrgeting) . AMPs surgeting) . Based oE. faecium, including persister cells and biofilms. The design strategy adopted here with charge and hydrophobicity balance could serve as a roadmap for developing AMPs that are bacterial selective. The lead peptide, 5L, exhibited narrow-spectrum activity, high antimicrobial potency, low mammalian cell cytotoxicity, and membrane-activating mechanisms, making it an attractive therapeutic candidate. Transcription-based mechanism assays and in vivo testing in higher organisms will be needed before 5L can move on to the next phase of anti-infective drug development.In summary, we developed short LfcinB6 peptides with activity against


In order to protect ferrite nanoparticles from oxidation and agglomeration, and to manufacture a novel catalytic system of anchored copper on the magnetic substrate, the Fe3O4 was core-shelled by adding tetraethyl orthosilicate. Next, the prepared Fe3O4@SiO2 was supported by phosphomolybdic acid (PMA) as the second layer of nanocomposite at 80 °C in 30 h. Eventually, the new nanocomposite of Fe3O4@SiO2-PMA-Cu was successfully synthesized by adding copper (II) chloride solution and solid potassium borohydride. The structure of magnetic nanocatalyst was acknowledged through different techniques such as EDS, VSM, XRD, TEM, FT-IR, XPS, TGA, BET and FESEM. The synthesis of β-thiolo/benzyl-1,2,3-triazoles from various thiiranes, terminal alkynes and sodium azide was catalyzed by Fe3O4@SiO2-PMA-Cu nanocomposite in aqueous medium. In order to obtain the optimum condition, the effects of reaction time, temperature, catalyst amount and solvent were gauged. The recycled catalyst was used for several consecutive runs without any loss of activity.The magnetic nanoparticles of Fe From view point of green chemistry, these reactions are of particular importance due to the significant reduction in the amount of solvents and energy required to separate and purify the intermediates. Moreover, MCRs play considerable roles in various research fields such as synthetic organic, biomedical, industrial chemistry, pharmaceutical and drug discovery research5.The Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes has been identified as click chemistry. Click reactions which offer high yield products in a short reaction time under mild conditions are fast, simple to use, easy to purify, eco-friendly, and regiospecific. The Huisgen 1,3-dipolar cycloaddition has found utilizations in a vast diversity of research areas, including pharmaceutical sciences, organic synthesis, and polymer chemistry7, antibacterial8, antitubercular9, analgesic10, local anaesthetic11, anticancer12, anti HIV13, antifungal14, antiallergic15, anticonvulsant16, antiproliferative17, antiviral, antioxidants18, antimalarial19 and anti-inflammatory20.1,2,3-Triazoles are a very valuable class of heterocycles that have received special attention in recent decades because of their biological and pharmacological activities such as antimicrobialβ-hydroxy-1,2,3-triazoles from azides, epoxides and terminal alkynes has been reported in the presence of different copper catalysts30. The strained three-membered heterocycles such as epoxides, thiiranes and aziridines are easily ring opened by various nucleophiles such as azides and amines. However, the synthesis of 1,2,3- triazoles from epoxies has been studied in a limited way using azides and alkynes, and the three-membered thiiranes have not been used except in one case recently reported31.In recent years, one-pot click synthesis of 34 and modern catalysis research35. The ferrite-based nanoparticles have been widely used as catalysts to perform various multicomponent reactions37 and organic syntheses42. Compared to the general separation, magnetic separation has appeared as a powerful, efficient, easy and rapid separation method47.Recently, ferrite nanoparticles due to recovery and reusability have received great attention in biomedicine49. In order to stabilize them against oxidation, corrosion and aggregation, their surface is modified with organic or inorganic materials. The surface of nano-ferrites could be improved by means of different materials such as polymers, bio molecules, silica, metals, metal oxides, surfactant, etc.50.Although nano-ferrites are promising class of the catalysts, but their surfaces are hydrophobic with very potent magnetic attractions and high surface to volume ratio, and they always suffer from adsorption troubles due to their high potential of self-aggregation. On the other hand, they are involved in other problems such as low quantity of functional groups on the surface, which disrupts their performance51. Phosphomolybdic acid (PMA) is one of the well-known polyoxometalates, which is commercially available and affordable52. But, the use of PMA is restricted because of its disadvantages including small surface area, low stability, dissolution effects53 and destruction in aqueous media54. Moreover, it has a high miscibility in polar solvents, which limits its application so it is very difficult to separate and recover55.Polyoxometalates (POM) are composed of transition metal oxide anions of molybdenum, tungsten and vanadium. This category of compounds bond small species of metal oxides and bulk solid oxometalates. Their exclusive attributes such as vast physical and chemical adaptability, optical and acidic activities as well as reducing and oxidizing capabilities turn them into the excellent compounds for research on the metal oxide activities56, positively charged polymer chains57, metal cations 60, and carbon61. The PMA properties such as high solubility, strong acidity and high negative charge, facilitates its anchoring on any support in various solvents59.In order to prevail these obstacles, PMA is embedded on useful support materials such as silica68, herein we wish to introduce Fe3O4@SiO2-PMA-Cu nanocomposite as an efficient and novel catalyst, whose PMA clusters are found to be anchored on Fe3O4@SiO2 surface, and copper nanoparticles are supported on the PMA surface. The new synthesized nanocomposite was utilized in three-component synthesis of β-thiol-1,4-disubstituted-1,2,3 triazoles from sodium azide, thiiranes, and terminal alkynes as a green and recyclable magnetic catalyst  have unique structures with a wide range of coordination positions comprising oxygen atoms, which are appropriate for anchoring the single atoms such as copper particles70. Since there are several possible coordination sites on the surface of PMA, it was selected as a support to trap the single metal atoms of copper in this study. The use of atomic catalysts leads to saving the quantity and cost of precious metals since they increase the efficiency and activity of the catalyst dramatically.Supporting polyoxometalates onto solid materials and decorating them with suitable porous supports such as metal oxides and MNPs is one of the most effective methods to improve their performance, which is achieved by increasing their active centers and reusability of these heterogeneous materials3O4@SiO2-PMA-Cu were synthesized in a four-step procedure . Then, PMA was added to a suspension of Fe3O4@SiO2 in ethanol, while being dispersed by sonication. In order to synthesize Fe3O4@SiO2-PMA-Cu, the prepared particles of Fe3O4@SiO2-PMA were added to a solution of CuCl2⋅2H2O in water and then the KBH4 powder was gradually added, while the mixture was strongly being stirred. Eventually, the dark brick-red sediment of Fe3O4@SiO2-PMA-Cu was separated magnetically, and then washed with distilled water, and dried at room temperature under air atmosphere.The nanoparticles of Fe3O4@SiO2-PMA-Cu nanocatalyst.The different techniques such as FT-IR, X-ray diffraction (XRD), energy dispersive X-ray spectrometer (EDS), field emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), vibration sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS), thermogravimetric (TG), Brunauer–Emmett–Teller (BET) and inductively coupled plasma optical emission spectrometry (ICP-OES) analyses were applied for characterization of new synthesized Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-PMA and Fe3O4@SiO2-PMA-Cu. As it is revealed in Fig. −1 and hysteresis phenomenon was not found. The magnetization curve quickly rises without showing any remanence or coercivity, and the sample displays a typical superparamagnetic behavior of soft magnetic materials at room temperature. The superparamagnetic property of these nanoparticles is a vital feature in their application because it prevents accumulation and aggregation of particles and enables them to re-disperse in the absence of a magnetic field immediately. The saturation magnetization (Ms) amount of the Fe3O4@SiO2-PMA-Cu MNPs was appropriate and the separation of the catalyst nanoparticles was easily carried out by using an external magnet.To confirm magnetic property of the synthesized nanocatalyst, VSM analysis was carried out. Figure 3O4, Fe3O4@SiO2, PMA, Fe3O4@SiO2-PMA and Fe3O4@SiO2-PMA-Cu. The absorption peaks at 3449 and 3358 cm−1 are assigned to the stretching vibration of H2O molecules and indicates the OH groups on the surface of the magnetic nanoparticles and hydroxyl groups in PMA structure respectively. The band around 1627 cm−1 corresponds to the bending mode of H2O molecules. The presence of SiO2 is confirmed by stretching and bending vibrations of Si–O. The absorption bands at 803 and 1097 cm−1 are related to Si–O–Si stretching vibrations, and the band observed at 478 cm−1 belongs to the bending vibration of SiO267. The peak at 950 cm−1 belongs to the Mo–O stretching vibrations, which confirms the existence of phosphomolybdic acid71. The formation of Fe3O4 nanoparticles was confirmed by the absorption band at 561 cm−1 which corresponds to the vibration of metal oxide bonds (Fe–O). A band appearing at 478.49 cm−1 is related to the stretching vibrations of Cu–O.Figure 3O4, Fe3O4@SiO2, PMA, Cu and Fe3O4@SiO2-PMA-Cu nanoparticles are shown in Fig. 3O4@SiO2-PMA-Cu is amorphous due to the coating of the ferrite surface with layers of silica, phosphomolybdic acid and copper nanoparticles, and the wide peaks confirm the small size of the particles at the nanoscale. In the XRD pattern of nanocomposite, all the peaks of Fe3O4, SiO2, PMA and Cu nanoparticles are detectable. The lines (220), (311), (400), (422), (511), (440), (620) and (533) related to 2Ө = 32.88, 35.64°, 44.11°, 53.43°, 57.60°, 64.01°, 71.95° and 75.01° respectively, are assigned to the diffraction of Fe3O4 crystals. These peaks are compatible with the standard data (JCPDS: 00–43-0317)72. The significant diffraction peaks are observed in case of PMA at 2Ө = 8.95°, 18.49°, 26.43°, 27.65° and 28.84° corresponding to (011), (202), (141), (311) and (312) crystallographic planes respectively (JCPDS: 00–043-0317)73. The broad peak at 2Ө = 22.90° is related to the amorphous SiO2 shell on the surface of Fe3O467. The copper diffraction peaks were compared with the standard sample (JCPDS 04–0836), and the peaks appearing at 2Ө = 43.71°, 50.70°, and 74.32° corresponding to the (111), (200), and (220) planes respectively, revealed the excellent coordination of the synthesized sample with its standard sample31.The X-ray diffraction (XRD) patterns of Fe3O4@SiO2-PMA-Cu nanocomposite are shown in Fig. 3O4 nanoparticles were synthesized at the nanoscale and coated with a dark gray silica layer, and the silica layer was entirely coated with phosphomolybdic acid. The PMA layer is visible in light gray. The TEM images also display that very small spherical Cu nanoparticles have been successfully deposited on the PMA layer, and they have completely surrounded the outer surface of the catalyst.TEM and FESEM techniques were used to determine the morphology and size distribution of the nanocatalysts. TEM images of the Fe3O4@SiO2-PMA-Cu that approve the formation of nancomposite. Small amounts of agglomerates were observed in the Fe3O4@SiO2-PMA-Cu surface due to the modification of the catalyst surface with non-magnetic layers and decreased magnetic properties. The information obtained from the FESEM images is consistent with the XRD and TEM data.Figure The chemical composition and percentage of nanocomposite elements were acknowledged using EDS data and elemental mapping patterns Fig. . In this3O4, Fe3O4@SiO2 and Fe3O4@SiO2-PMA was also investigated using EDS technique, and the results were presented in Fig. The elemental composition of catalyst intermediates Fe3O4@SiO2-PMA-Cu, Fe3O4 and Fe3O4@SiO2 nanoparticles are shown in Fig. 3O4@SiO2-PMA-Cu. The photoelectron lines representing Cu 2p, Fe 2p, O 1 s, Mo 3d, P 2p and Si 2p at 920, 725/710, 533, 232, 130 and 106 eV respectively can be observed in the survey spectrum of nanocatalyst74. The peaks of Fe element for Fe3O4@SiO2-PMA-Cu and Fe3O4@SiO2 were weakened, indicating that Fe3O4 core was covered by SiO2 shell. The results agreed well with EDS data.The chemical composition of the different nanoparticles was also characterized using XPS technique. The XPS survey spectra of Fe3O4@SiO2-PMA-Cu, thermal gravimetric analysis (TGA) was carried out in the temperature range of 70–800 °C under nitrogen atmosphere , indicating that Fe3O4@SiO2-PMA-Cu catalyst is mesoporous.The NH-1,2,3-triazol-1-yl)ethane-1-thiol was optimized under different conditions. The various empirical factors such as temperature, catalyst quantity, solvent, reaction time and the amount of reactants were examined, and the acquired results were provided in Table 3O4@SiO2-PMA-Cu (0.1 g) as catalyst in water at 55 °C (Table 3O4@SiO2-PMA-Cu even after 10 h (entry 1). The catalyst amount was optimized using different quantities of Fe3O4@SiO2-PMA-Cu nanocomposite , and 0.1 g of catalyst gave the eligible outcome. The product yield and reaction time were strongly influenced by the concentration of catalyst, so that the product yield and reaction rate increased dramatically by increasing the amount of catalyst from 0.05 to 0.1 g (entries 2–4). The higher amount of catalyst had no effect on the product yield (entry 5). The turn over number (TON) and turn over frequency (TOF) of the present catalyst were also calculated for the model reaction based on the amount of the active metal used (Cu) and they were found to be 1214 and 346 h−1 respectively.The reaction of styrene episulfide, sodium azide and phenyl acetylene was chosen as model reaction, and the synthesis of 2-phenyl-2-. Water as a green and eco-friendly solvent was the most privileged choice because the yield of the product in water was higher than all other solvents (entry 4).The effect of various polar and non-polar solvents on reaction was examined. The polar solvents such as HIn order to investigate the effect of temperature, the reaction was performed at different temperatures. The reaction result was not desirable at room temperature (25 °C) and the product yield was low after 9 h (entry 14). As a result of raising the temperature to 45 °C, the experimental data improved and the product yield increased by 72%, and the reaction time was reduced to 4 h (entry 15). Further raising the temperature to 55 °C significantly improved the product yield as well as reduced the reaction time (entry 4).H-1,2,3-triazol-1-yl)ethane-1-thiol, the reaction was evaluated separately using Fe3O4, SiO2, PMA, Cu, Fe3O4@SiO2 and Fe3O4@SiO2-PMA under the optimal conditions 75. In both paths, Fe3O4@SiO2-PMA-Cu plays the role of catalyst  is more stable; however, the regioselective ring opening of thiiranes with alkyl and allyl groups is carried out from the less hindered carbon via SN2 type of mechanism (β-cleavage). In pathway A, in order to confirm the catalytic role of Fe3O4@SiO2-PMA-Cu in the preparation of 2-azido-2-arylethanthiol from styrene episulfide and sodium azide, the reaction was performed in the absence of catalyst, and only a very small amount of ring opened product was produced. During the reaction, gas chromatography (GC) and thin layer chromatography (TLC) runs of the reaction mixture were utilized to monitor the consumption of styrene episulfide and sodium azide and the formation of 2-azido-2-phenylethanthiol intermediate. FT-IR spectrum was used to characterize 2-azido-2-arylethanthiol through stretching frequency of 2097 cm−1 corresponding to the azide 76.The specific catalytic property of Fe3O4@SiO2-PMA-Cu is due to synergistic effect of all components. The formation of intermediate (I) was confirmed by characteristic absorption peak of 3293 cm−1 for phenylacetylene, indicating that terminal hydrogen atoms had not been removed during the activation process  was produced after the release of catalyst nanoparticles.The proposed mechanism for synthesis of yst Fig. . First, ide Fig. a. In patess Fig. b. Interm3O4@SiO2-PMA-Cu magnetic nanocomposite was described. The new synthesized nanoparticles were characterized using various techniques such as FT-IR, XRD, VSM, EDS, XPS, TGA, BET, TEMand FESEM. Then, it was utilized as a practical catalyst for one-pot synthesis of β-thiol-1,4-disubstituted-1,2,3-triazoles from sodium azide, terminal alkynes, and diverse thiiranes in water. The synthesis of 1,2,3-triazole from thiiranes has not been reported so far, except in one case. The mentioned protocol offers several advantages such as reusability and easy separation of the heterogeneous magnetic catalyst, perfect regioselectivity, short reaction times, high product yields, utilizing of the green solvent and simple work-up procedure.In this research, the synthesis of novel, efficient, robust and reusable Feα radiation, λ = 1.54056 Å). Magnetic property of nanocatalyst was evaluated by VSM  at room temperature. The amount of copper in the catalyst was measured by Perkin Elmer Optima 7300DV ICP-OES analyzer. IR spectra of the catalyst and triazole products were recorded on Thermo Nicolet Nexus 670 FT-IR. 1H/13C NMR spectra of products were recorded on a Brucker 500 MHz spectrometer in deuteriochloroform with tetramethylsilane as internal standard. HRMS analyses were carried out in the electron impact mode (EI) at 70 eV. Melting points were also determined on an Electrothermal IA9100 microscopic digital melting point device. Brunauer–Emmett–Teller (BET) surface areas were measured by nitrogen adsorption at 77.35 K on Belsorp-max, BEL Japan. The electronic states of the samples were analyzed using X-ray photoelectron spectroscopy . thermogravimetric analysis (TGA) was determined on Perkin Elmer, Diamond TG/DTA.All reagents and substrates were bought with high quality without any need for further purification from Aldrich  and Merck  Chemical Companies. The prepared nanocatalyst was identified by TEM images recorded using an EM10C-100 kV series microscope from Zeiss Company (Germany). FESEM images were obtained employing FESEM-TESCAN. The elemental composition study was performed using the energy dispersive X-ray spectrometer (EDS) analysis on a MIRA3 FE-SEM microscope , equipped with an EDS detector . The XRD intensities were measured on a Bruker D8-Advanced diffractometer , FeCl3·6H2O , NaOH , and NaCl  were blended in a mortar and then were milled for 50 min. The beginning of the reaction was accompanied by the release of heat. After a few minutes, the dark brown paste was obtained. The excess salt of the resulting mixture was removed through repeated rinsing with distilled water. The obtained product was placed in oven at 80 °C for 2 h, and then purified by calcination at 700 °C for 1.5 h to produce the dark brown powder of Fe3O4 MMPs.Fe3O4@SiO2 were synthesized through an improved sol–gel method67. In a typical procedure, 0.5 g Fe3O4 MNPs was added to a mixed solution of ethanol (50 mL), distilled water (10 mL) and NH3·H2O  and dispersed using ultrasonication for 1.5 h. Then 1.5 mL TEOS (tetraethyl orthosilicate) was added dropwise to the above mixture and sonicated for a further 15 min. After ongoing stirring at room temperature for 24 h, the yellow Fe3O4@SiO2 MNPs were accumulated, repeatedly washed with double distilled water and dried in an oven at 100 °C for 22 h.Silica coated MNPs of Fe3O4@SiO2 nanoparticles, Fe3O4@SiO2 (0.5 g) was first suspended in ethanol (20 mL) and then dispersed by ultrasound for 30 min. Next, the solution of PMA (0.5 g) in ethanol (5 mL) was prepared and gently added dropwise to the suspension. The resulting brick-red mixture was stirred for 30 h under inert atmosphere of nitrogen while heating at 80 °C. The magnetic nanocomposite of Fe3O4@SiO2-PMA was collected by a magnet, washed several times with distilled water and dried at 60 °C for 20 h. The Fe3O4@SiO2-PMA nanocomposite was obtained as a soft brick-red powder.In order to synthesize the phosphomolybdic acid (PMA) immobilized on the surface of Fe3O4@SiO2-PMA, first, CuCl2·2H2O  was dissolved in distilled water (50 mL) and then 1 g Fe3O4@SiO2-PMA was added to it and the mixture was stirred vigorously for 30 min. Next, 0.1 g potassium borohydride powder (KBH4) was gradually added to the mixture at room temperature and stirred for 1 h to reduce Cu2+ cations to copper nanoparticles. The obtained Fe3O4@SiO2-PMA-Cu nanocomposite was washed three times with double distilled water and after drying under air atmosphere, it was collected as a dark brick-red powder.To anchor the copper nanoparticles on the Fe77. In a typical experiment, a mixture of epoxide (1 mmol) and alumina supported thiourea  was placed in an agate mortar and milled for the required time at room temperature. The reaction completion was tracked by TLC utilizing n-hexane/ethylacetate (10:2) eluent. After the reaction was finished, the contents of the mortar were washed with ethylacetate and then filtered. The solvent containing thiirane was evaporated to produce the pure thiirane as a pale yellow oil.Different thiiranes bearing electron-donating or -withdrawing substituents were synthesized using previously reported procedure3  and H2O (5 mL) in a round-bottomed flask, equipped with a magnetic stirrer and condenser, 0.1 g of Fe3O4@SiO2-PMA-Cu nanocomposite was added. The mixture was stirred at 55 ºC for an appropriate time period (1.5–3.5 h). The reaction process was tracked using TLC and n-hexane/ethylacetate (10:2) eluent solvent. After the reaction ended, the Fe3O4@SiO2-PMA-Cu nanoparticles were accumulated utilizing an external magnet and reused in the consecutive cycle. After extraction of the aqueous layer with ethyl acetate and drying over anhydrous sodium sulfate, the organic solvent was evaporated under vacuum and the crude 1,2,3-triazoles were produced. The obtained products were recrystallized with EtOH/H2O (1:1) to give the pure β-thiol-1,4-disubstituted-1,2,3-triazoles in 85–98% yield , terminal alkyne (1 mmol), NaN3O4@SiO2-PMA-Cu were reused in the next cycle without any significant loss of catalytic activity or magnetic properties.In order to recycle the catalyst, first, magnetic nanoparticles were collected utilizing an external magnet, washed three times with ethyl acetate and distilled water, and dried under air atmosphere. The dried nanoparticles of FeSupplementary Information.