Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants  to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.  Cytochrome P450s (P450s) comprise a huge superfamily of ubiquitous heme-thiolate enzymes. Despite sharing a common fold, P450s catalyze different types of regio-, chemo-, and stereospecific reactions on a vast variety of structurally unrelated compounds ,2.Notwithstanding the wide substrate versatility, substrate binding to P450s is a finely regulated event. Regulation is achieved through enzyme-substrate interactions established within the active site that often activate conformational transitions. The structural dynamics of P450s mainly consist of the rearrangements of specific secondary structural elements that close the active site around the substrate, promoting the displacement of the axial water ligand from the heme iron that switches to the high spin-state, and minimizing the access to the external environment . ProductStreptomyces antibioticus, is the P450 involved in the polyketide biosynthesis of the antibiotic oleandomycin  and [S0] are the enzyme and the substrate analytical concentrations, respectively. All of the binding experiments were performed in duplicate.The dissociation constant  after each substrate addition, and the appropriate blank was subtracted.All of the spectral determinations were made using a Jasco V-650 spectrophotometer controlled by a Jasco programmable Peltier element (EHC-716) and a HP-8453 spectrophotometer equipped with a Peltier temperature control accessory (HP-S9090A).Purified E89Y, G92W, and S240Y OleP mutants  were co-crystallized at a concentration of 19 mg/mL (0.45 mM) with 6DEB at saturating concentration (1 mM), using the hanging-drop vapor-diffusion method at 293 K. Crystals of the three mutants were obtained in sodium formate, as detailed in v/v glycerol prior to flash-freezing in liquid nitrogen, while S240Y-6DEB crystals were frozen without the cryo-protectant.Crystals of E89Y-6DEB and G92W-6DEB were mounted on nylon loops and cryoprotected by immersion in a solution containing the crystallization buffer supplemented with 20% X-ray diffraction data were collected at 100 K on a PILATUS detector  at ELETTRA synchrotron . Data were indexed, scaled, and integrated using XDS package  and Aimlo|–|Fc| map contoured at ±3 σ and the 2|Fo|–|Fc| at ±1 σ. Five percent of the reflections were excluded from refinement and used for the Rfree calculation ..20].DK are reported in Therefore, the change in heme absorption was measured at 298 K as a function of substrate concentration for all of the mutants, while the enzyme concentration remained fixed. The observed binding curves were satisfactorily fitted with a hyperbolic equation, the estimated DK values revealed some increase in 6DEB affinity with respect to the wild type.Similarly to the wild type, binding to E89Y causes a type I spectral change. This mutation marginally alters the affinity for the aglycone, but it halves the amplitude of the spectral transition, thus indicating the presence at the equilibrium of a minor population of enzyme, which is converted to the high spin state by 6DEB binding with respect to OleP wild type . BindingTo analyze how mutations are accommodated in the OleP active site, we determined the crystallographic structures of E89Y, G92W, and S240Y in complex with 6DEB.Crystals were obtained in the C2 space group, diffracted at resolutions between 2.1 and 2.7 Å, and contained six monomers in the asymmetric unit.The overall structure of the complex of 6DEB with mutants resembles the wild type complex obtained under similar crystallization conditions, characterized by high ionic strength . In all All of the structural features typical of the wild type OleP bound to 6DEB in the closed state are maintained in the mutants. More in details, (i) the closure of the active site involves a pronounced structural reorganization of the F, G, and I helices, of the HI loop over the heme pocket and less pronounced of the BC loop if compared to OleP-6DEB in the open state ; (ii) thTherefore, in the presence of E89Y, G92W, and S240Y mutations, OleP still binds 6DEB and, in the crystallization conditions explored, it adopts a closed and catalytically competent conformation.The presence of the E89Y, G92W, and S240Y mutations in the OleP active site has been initially assessed by calculating the omit map for the relative residues. A clear bulkier electron density, in correspondence of the mutated sites, was observed.3 in the wild type and ~582.5 and ~566.8 Å3 in G92W and S240Y, respectively  between the C5-OH of 6DEB and the aromatic ring of Y240 e. In thiThe insertion of a tryptophan in position 92 and of a tyrosine in position 240 also generate additional contacts within the protein structure, involving residues located in the active site of the enzyme. In G92W, we identified: (i) A T-stacking interaction between W92 and F84 (BC loop) and F296 (β-hairpin β3); (ii) an induced dipole-dipole type interaction between the aromatic ring of W92 and the δ-carbonyl group of N236 and the ε-amino group of Q193 d. In S24In order to analyze the effect of mutations on the conformational dynamics of the closed-bound OleP, MD simulations were performed on the wild type OleP (pdb code: 5MNS) , E89Y, aDifferences in the accessible conformational space between the two OleP mutants and the wild type emerge from the essential dynamics analysis of the concatenated trajectory. In fact, the projections of the three trajectories on the essential subspace—as defined by the first two eigenvectors—clearly show that WT, G92W, and E89Y explore different regions of this subspace . In partInterestingly, the analysis of the eigenvalues associated to the calculated eigenvectors shows that the first two eigenvectors describe ~64% of the total variance observed along the MD trajectories, suggesting that they are able to properly describe the main protein motions , 131–158 (helices D and E), and 209–242 .The local analysis of the protein cavities as obtained by MD simulations show that in the G92W mutant the distance distribution maximum between the heme-iron and substrate is slightly higher than the WT and E89Y mutant . This miThese results highlight the relevant effects on the structural-dynamical behavior due to single mutations, which are able to shift the accessible conformational space, at both local and global level, within the ns time-scale.The mechanism that elicits the productive structural response of a P450 upon substrate binding is characteristic of each specific member of the family and it depends on each enzyme’s intrinsic structural plasticity and on the nature of the physiological substrate. Its full understanding is very relevant in the perspective of rationally exploiting the unique and versatile catalytic properties of each P450 on alternative compounds for diverse biotechnological applications ,32,33.In this work, we analyzed the case of the bacterial cytochrome P450 OleP. The substrate versatility of this enzyme, that naturally catalyzes the epoxidation of aliphatic carbons of macrolidic substrates, has lately attracted the pharmaceutical research attention given its capability to hydroxylate steroid compounds ,35,36. MGiven the emerging interest towards the OleP enzymatic activity, in this study we have extended the characterization of the substrate binding properties of this P450, analyzing the role of a small polar cavity of the active site of the protein (the solvent cavity), that forms upon OleP closure, in the substrate binding process and in the subsequent structural rearrangements that lead to the productive substrate-bound state. We produced three OleP mutants to introduce a steric perturbation to the cavity. We substituted residues lining the cavity, namely E89, G92 (BC loop), and S240 (I helix), with bulky amino acids to fill its volume.The crystal structure of the mutants, obtained in conditions that are known to stabilize the closed conformation of OleP, i.e., high ionic strength, confirmed that substitutions at G92 and S240 with a tryptophan and a tyrosine, respectively, occupied the cavity. Within the solvent cavity, the aromatic side chain of S240Y and G92W form additional direct and directional contacts with the substrate and with other residues within the distal pocket, inducing the partial or the total removal of solvent molecules from that region. Conversely, E89Y mutation directs the amino acid side chain towards the outer environment, increasing the empty volume, as shown by both crystal structure and MD simulation analyses. Equilibrium binding experiments with 6DEB performed on the latter mutant showed only a slight decrease in the binding capacity with respect to the wild type enzyme. On the contrary, the presence of G92W and S240Y mutations alters the binding properties of OleP to 6DEB. Indeed, the type I spectral change observed upon 6DEB binding, is converted to a type II shift of the UV-visible heme Soret peak, with some increase in affinity. This spectroscopic behavior is typical of a ligand that upon binding induces the stabilization of the hexacoordinate low spin state of the P450 heme iron. This, in our system, might correspond to the stabilization of a complex in which 6DEB is too distant from the heme iron to induce the displacement of the sixth coordinating water molecule, whose strong ligand field appears further enhanced by substrate binding. This state in OleP has been previously described as the open-bound form . On the In light of this evidence and with the aim to demonstrate an effect of G92W and S240Y mutant on the structural dynamics of OleP, we examined the change of the UV-visible spectra of all OleP mutants bound to 6DEB as a function of sodium formate concentration . IncreasNotably, the heme reactivity response to the change of ionic strength conditions depends on the mutation. Indeed, while the effect of the ionic strength on the spin state equilibrium of the wild type and E89Y is comparable, showing the total low-to-high spin shift at I = 2.5–3 M, in G92W and S240Y mutants the shift is not complete even at the highest ionic strength conditions tested (I = 3.5 M). In particular, the S240Y mutant bound to 6DEB only starts shifting the equilibrium towards the high spin at I ≥ 3 M. The different degree of response to ionic strength conditions may arise from a different effect of the mutations at position 92 and 240 on the structural dynamics of the protein. In agreement with this observation, the MD simulations performed on the WT, E89Y, and G92W closed complex clearly show that the effect of the mutations is to alter their accessible conformational space. Indeed, the principal components analysis on the MD trajectories revealed that the regions sampled in the common essential subspace of the three OleP forms are different. In particular, the first eigenvector, i.e., the principal motion direction, can discriminate between WT and the two mutants, whereas the second eigenvector is able to further discriminate between the G92W and the wild type and E89Y complexes (the eigenvector components as well as their associated eigenvalues are reported in This qualitative structural analysis suggests that G92W and S240Y mutations may constitute a wedge that alters the intrinsic flexibility of OleP, increasing the free energy barrier for the open-to-closed transition. Therefore, at physiological ionic strength conditions, substrate binding to G92W and S240Y mutants cannot trigger the structural transition that induces the displacement of the water molecule from the sixth coordination position of the iron and the spin-state shift, not even on a partial population of the enzyme, as it occurs in the case of wild type and E89Y variants . We cannBacillus megaterium, where bulkier mutations at a site topologically homologous to the solvent cavity of OleP enhanced substrate binding affinity and the catalytic efficiency of the enzyme towards fatty acid substrates [All considered, our data suggest that filling the solvent cavity with bulky residues is not a viable strategy in future engineering work on the P450 OleP. The consequent alteration of the conformational dynamics, that emerges from the data herein discussed, seems to induce the accumulation at equilibrium of the non-productive form of the enzyme-substrate complex. This is in contrast to other P450 enzymes, such as the CYP102A1 from bstrates . As a fuIn conclusion, our data point to a role for the solvent cavity as the pivot of the OleP dynamics. In the perspective of further engineering the P450 OleP for future sustainable enzymatic production processes, it must be considered that the intrinsic flexibility of this site must be preserved to allow for the proper protein movements, which are productively triggered by substrate binding.


The structures of the synthesized compounds were confirmed by nuclear magnetic resonance spectroscopy (NMR), electrospray ionization high-resolution mass spectrometry (ESI HRMS), and tandem mass spectrometry methods (HCD MS/MS). Three different derivatives of 2,6-bis((benzoyl-R)amino)pyridine were used as carriers in membrane processes and as extractants in classic solvent extraction. In each case, the single derivative recovery was carried out on a model solution that contained only one type of metal ions. Spectrophotometry studies were performed to determine the stability constants of the complexes formed by the synthesized species with analyzed metals ions. The results obtained indicate that the synthesized compounds form stable complexes with Cu(II), Ni(II), Co(II), and Zn(II) ions and can be used in both types of studied recovery processes. However, the effectiveness of the synthesized compounds in the recovery of metal ions depends both on the structure of compounds and properties of metals as well as on their concentration.In this paper, the application of new substituted 2,6-bis((benzoyl-R)amino)pyridine (R = H, 4-Me, and 4-NMe Regardless of their origin, the most intensive signal on the spectrum  derivatives are similar, regardless of the differences in their structure. Some generated products are the result of a simple breaking of molecular bonds , other fragments originate from more complex intramolecular rearrangements, which are typical for HCD MS/MS . While a detailed analysis of the fragmentation processes was not the subject of this work, the results of the higher energy collisional dissociation experiments performed for the singly charged protonated ions of the A1–A3 compounds make it possible to confirm the structures of the species analyzed.The results of the tandem mass spectrometry experiments performed indicate that the main HCD dissociation processes of all 2,6-bis((benzoyl-R)amino)pyridine  of the created complexes were calculated using the known method .1, log K2 were assigned to stability constants of complexes, in which the molar ratio of ligand:metal ions was 1:1 and 1:2, respectively.The determined values of the stability constants (log K) of complexes are shown in The higher value of the stability constant (log K > 1.0) indicates that at equilibrium, the activity of the complex is larger than the product of activities of the metal ion and ligand (Equation (1)).As a result, the high value of log K indicates that the ligand binds to the metal ion more strongly than water molecules. The stability constant is then used as a measure of the thermodynamic stability of the complex. An increase in the number of ligands in the coordination sphere usually leads to a decrease in the number of water molecules to be replaced. This is why in some cases, it is found that complexes with a larger number of ligands are more stable than others because of unusual structural changes and differences in the electronic configuration of the metal ion. The complexes with a higher crystal field stabilization energy value will be stable, and the stability constant for those complexes will be high .1 for complexes with metal ions such as cobalt and nickel has a value amount to 5.Due to their structure  the tested derivatives belong to the so-called Lewis bases. Such compounds easily react with metal ions—the so-called Lewis acids. Therefore, in aqueous solutions, complexes of the tested derivatives with selected metal ions are characterized by quite stable structures. They can be compared with the stability of complexes formed by β-diketones, whose log K3)2 groups with the chosen metal ions were the highest. This compound created the most stable complexes, especially with copper(II) and nickel(II) ions. This may be explained by a substituent effect of the –N(CH3)2 group in the deprotonated ligand. The electron donating –N(CH3)2 moiety increased the basic character of the (O–C–N–C–N)—making it more potent to interaction with the Lewis acid.The stability constants of complexes of a derivative with the –N derivatives as extractants. The experiments were carried out for solutions in which the ligands and metal ions concentration ratios were 1:1, 1:2, and 1:5, respectively. The extraction percentage (M%E) of the metal ions is described by the following equation:MD is the division ratio determined experimentally; aqV is the volume of the aqueous phase [dm3]; orgV is the volume of the organic phase [dm3] .The solvent extraction of copper(II), nickel(II), cobalt(II), and zinc(II) ions was performed with 2,6-bis((benzoyl-R)amino)pyridine  to the sum of the concentrations of all the substances in the aqueous phase (Σ[M]aq).The division ratio (Equation (3)) is the ratio of the sum of the concentrations of all the substances in the organic phase  ions and the lowest for Co(II).The obtained division ratios  for the The percent of the extraction of metal ions with all of the investigated extractants is shown in the plots below.M was more than 90% for nickel(II), cobalt(II), and zinc(II) with all A1, A2, and A3 ligands. However, those ligands were not as effective during copper ion extraction , Ni(II), Co(II), or Zn(II) ions, respectively, the membrane extraction processes were started. Metals ions were adsorbed on the surfaces of the membranes. The ligand molecules  doped into the polymer membranes bound metals ions thanks to the complexation reactions.RF) was also determined (Equation (4)).c0 is the initial concentration of metal ions in the feed phase [mol/dm3] and c is the concentration of metal ions after time t in the feed phase [mol/dm3].After 24 h of membrane extraction process, the percentage of metal ion removal from the solutions (%The results of the membrane extraction processes performed are presented in tq is the sorption capacity (mg/g); V is the volume of the solution (dm3); m is the mass of the sorbent (g), and ic and tc are the analytical metal ion concentrations in the solution at the beginning and after an appropriate time of the sorption process (mol/dm3), respectively.The analysis of the metal ion sorption process onto the membranes with 20 wt.% of the investigated compounds used as a carrier was carried out using Equation (5):The sorption capacity of the membrane with all of the ligands A1–A3 as carriers after 24 h of the sorption of investigated metal ions is presented in t of investigated membranes was obtained for Zn(II) and Co(II) ions sorption and was equal to 3.81 mg/g and 3.42 mg/g, respectively. The lowest qt was observed for Cu(II) ions (0.17 mg/g). The received results can be compared with other literature data  and fly ash (FA) was 3.22–3.29 mg/g at 293 K [The highest qat 293 K , and theat 293 K .2) derivatives for the removal of metal ions (copper(II), nickel(II), cobalt(II), and zinc(II)) from aqueous solutions was described.In this study, the application of a series of new 2,6-bis((benzoyl-R)amino)pyridine benzoate, respectively. NMR and electrospray ionization high-resolution mass spectrometry (ESI HRMS) and higher energy collisional dissociation tandem mass spectrometry (HCD MS/MS) methods were successfully used to confirm the structures of all synthesized ligands. Given the high mass accuracy of HRMS mass spectrometry and a relatively easy and straightforward interpretation of the simple fragmentation spectra (HCD MS/MS) of the analyzed compounds, there is no doubt about the elemental composition, charge, or structure of the ions formed.The experiments of the complexation properties confirmed that the series of new compounds exhibited high complex-forming properties. The various molar ratio of ligand:metal was proved to form complex metal ion complexes with the investigated ligands. Application of the spectrometric method allowed us to determine the ratio of ligand to the metal in the studied complexes as well as their stability, which was quite high.Thanks to the complex-forming properties of the compounds A1–A3, it is possible to use synthesized compounds as very efficient extractants for removing metal ions from aqueous solutions. Studied derivatives can also be used as carriers in the membrane processes. However, to increase the efficiency of membrane processes, further research is needed, where membranes containing a different polymer and/or plasticizer will be used. Since the synthesis of chemical compounds used in this study as extractants and carriers is relatively easy and cheap, and these compounds have strong complexing properties, the results of this research may prove to be a new direction in the search for simple organic ligands to remove metal ions from waste.


An X-ray diffractometer was used to analyze the phase of the residue after quasi-static compression experiment. The results show that: (1) With the increase in molding parameters (preforming pressure and the pressure holding time), the compressive strength, failure strain and toughness of Al-rich Al/PTFE/TiH2 specimens first increase and then decrease. The best molding process parameters of Al-rich Al/PTFE/TiH2 materials are preforming pressure 240 MPa and the pressure holding time 100 s. (2) For unsintering specimens, when the preforming pressure is less than 150 MPa, the porosity of the specimen increases slowly at first and then decreases. When the preforming pressure is greater than 150 MPa, the porosity of the specimen increases first and then decreases. When the pressure holding time is no more than 100 s, the porosity of the specimen decreases gradually. When the pressure holding time is more than 100 s, the porosity of the specimen increases first and then decreases. For sintered specimens, when the preforming pressure is less than 100 MPa, the porosity of the specimen decreases gradually. When the preforming pressure is greater than 100 MPa, the porosity of the specimen first increases and then decreases. With the increase in the pressure holding time, the porosity first increases and then decreases. For each preforming pressure specimen, compared with that before sintering, the porosity after sintering either decreases or increases. For each the pressure holding time specimen, the porosity increases after sintering compared with that before sintering. The microstructure of PTFE crystal inside the specimen is mainly planar PTFE crystal. The size and number of planar PTFE crystals are significantly affected by the molding parameters, which further affects the mechanical properties of Al-rich Al/PTFE/TiH2 specimens. When the preforming pressure is less than 100 MPa, the planar PTFE crystals are small and few, which results in the worst mechanical properties of the specimens. When the preforming pressure is more than 100 MPa and does not contain 240 Mpa, the planar PTFE crystals are small and there are more of them, which results in better mechanical properties of the specimens. When the preforming pressure is 240 MPa, the planar PTFE crystals are large and numerous, which results in the best mechanical properties of the specimen. When the pressure holding time is 100 s, the planar PTFE crystals are large and there are more of them, which results in the best mechanical properties of the specimen. (3) The reactivity of Al-rich Al/PTFE/TiH2 specimens with TiH2 the content of 10% under quasi-static compression is not significantly affected by the molding parameters.Preforming pressure and the pressure holding time are important parameters of the molding process, which directly affect the mechanical properties of materials. In order to obtain the best molding parameters of Al-rich Al/PTFE/TiH Al/PTFE is a typical reaction material, which is impacted at high velocity to produce explosion light and release a lot of chemical energy ,2. The r2 is a high energy metal hydride with high hydrogen content. The melting point of TiH2 is 400 °C, and above this temperature, TiH2 decomposes slowly and dehydrogenates completely at 600–800 °C in vacuum. When the hydrogen content is 3.9%, the calorific value of unit mass released by TiH2 is 21.5 MJ/kg [2 and release a large amount of energy. At present, many scholars have introduced TiH2 into conventional energetic materials such as propellants and explosives, and have carried out a series of related properties research. Li [2 on the burning rate of propellants. It was found that the addition of TiH2 can increase the heat of propellant conduction from gas phase to combustion surface, thus increasing the burning rate of propellant. Cheng [2 to the emulsion explosive for underwater explosion experiment. The experimental results show that the total energy and specific impulse of the mixed explosive are obviously improved, and the explosive ferocity is higher than that of the emulsion explosive without TiH2. Some scholars [2 into the strong oxidant KClO4, and studied the compatibility and critical ignition temperature of the mixture. It was found that the decomposition of TiH2 in the strong oxidant was negligible, and the ignition temperature of KClO4 would not change due to the addition of TiH2.TiH.5 MJ/kg , which iarch. Li  studied t. Cheng  added Tischolars ,13,14 inf/ZL301 composite with 48% fiber volume fraction by vacuum assisted pressure infiltration method, and studied the effect of infiltration holding pressure time on Microstructure and mechanical properties of 3D-SiCf/Al composite. The results show that prolonging the pressure holding time can reduce the void and agglomeration in the composite fiber bundle, and the tensile strength of the composites increases first and then decreases with the increase in holding pressure time, which is due to the deterioration of the mechanical properties of the composites due to the strong interfacial reaction. Xia [2O3-TiC/Fe matrix composite ceramics reach the best value, the hardness is 467 HB, the bending strength is 882 MPa, the relative density is 99.6%, and the wear rate is 0.102%. Molding process is an important link in the preparation of material specimens, which directly affects the properties of material specimens. In the last decade, scholars have mainly focused on researching the molding process of non-active materials, and there are not many reports on this kind of research. Zhu  studied ion. Xia  Studied 2 into Al/PTFE energetic materials for the first time under zero oxygen balance, and mainly aimed to analyze the effect of different TiH2 content on the static mechanical properties of Al/PTFE/TiH2 composites under equilibrium condition. Through quasi-static compression experiments, the effects of different content of TiH2 on the mechanical properties and reaction characteristics of Al/PTFE were compared and analyzed. The results showed that when the content of TiH2 was 5%, the probability of reaction was 90% and the material strength reached the maximum value of 108 MPa, which was 15.1% higher than that of Al/PTFE material. Although the content of TiH2 has a significant effect on the mechanical properties and reaction characteristics of Al/PTFE active material, the jet formed by the Al/PTFE active cover under zero oxygen balance is easy to diverge, which is not conducive to the penetration and perforation of the target, while excessive Al can enhance the cohesiveness of the Al/PTFE active jet, thus improving the penetration effect of the target. At the same time, Yu did not study the effect of technology on the properties of Al/PTFE/TiH2 composites under equilibrium. Therefore, the preparation process, mechanical properties and reaction characteristics of Al-rich Al/PTFE/TiH2 active composites composed by adding TiH2 into Al/PTFE energetic materials with excess Al are still unclear. At present, no research papers on the technology of Al/PTFE/TiH2 active materials under equilibrium have been published at home and abroad. Based on the above, this paper starts from the molding process of the preparation process, the influence of six groups of preforming pressure parameter  and four groups of the pressure holding time parameter  on the mechanical properties and micromorphology of Al-rich Al/PTFE/TiH2 materials were analyzed by quasi-static compression experiments.Recently, Yu ,21 intro2: 4–6 μm ; Anhydrous ethanol: purity 95% . The physical and chemical properties of each component are listed in The main raw material powders are as follows: Polytetrafluoroethylene (PTFE): 27 μm ; Al: 6–7 μm ; TiH2F4 = 4AlF3 + 6C, it can be seen that when TiH2 is introduced into Al-rich Al/PTFE (wt50%/wt50%) active material, the maximum content of TiH2 is 32%. According to the reference [2 active material with TiH2 content of 10% has moderate energy release rate and reaction threshold. Therefore, In this paper, Al (wt40%)/PTFE (wt50%)/TiH2 (wt10%) was selected as the composition of 10 groups of specimens.According to the main reaction equation of the material: 4Al + 3Ceference ,21, the 2 is 2 mixed powder is as follows:Assuming that the volume fraction of PTFE powder is 2 is 2 is Assuming that the mass fraction of Al powder is 2 is as follows:According to Equations (1) and (2), the maximum theoretical density of Al-rich Al/PTFE/TiH1, pending specimen is suspended by a gravimeter and placed in a beaker with water and does not touch the wall of the beaker, the measured gravity is N, the volume of the pending specimen is V, the corresponding density of water at ambient temperature is ρwater, and the local acceleration of gravity is glocal. According to Archimedes’ principle [Assuming that the mass of the pending specimen in the air is mrinciple , then(4Combined with Equations (4) and (5), the actual density of the specimen is as follows:2 active specimens is as follows:2 powder are weighed by electronic scale and mixed in a beaker. While adding an appropriate amount of anhydrous ethanol into the beaker, the mixture is continuously stirred for about 30 min to make a “sesame paste”-like liquid, that is, the three materials are fully mixed. The mixed liquid was dried in a vacuum drying oven at 55 °C for 48 h, and the bulk Al-rich Al/PTFE/TiH2 solid mixture was obtained.Drying of mixture: according to the mass ratio of 40/50/10, Al powder, PTFE powder and TiH2 solid mixture is crushed with a glass rod, and continuously stirred to powder state, then put into a transparent bag, and then drop the transparent bag containing powder for at least 30 times to obtain uniform Al-rich Al/PTFE/TiH2 powder. The powder is pressed into cylindrical large cake specimens with different preforming pressure and holding time by hydraulic press and forming mold.Compaction: the block Al/PTFE/TiH2 specimen is obtained by machining. Some of the experimental specimens are shown in Sintering process: in the sintering furnace with argon atmosphere, evenly raise the temperature of columnar large round cake to 360 °C at the rate of 50 °C/h, holding for 6 h, then evenly lower the temperature to 315 °C at the rate of 50 °C/h, holding for 4 h, and then uniformly lower the temperature to room temperature at the rate of 50 °C/h. The sintering process curve is shown in The complete process of preparing Al-rich Al/PTFE/TiHThe experimental arrangement is shown in α radiation λ = 0.15416 nm), the scanning range 2θ was 10–90°, the scanning step was 0.02°, and the scanning speed was 4°/ min.The X-ray diffractometer (XRD) of Rigaku smartlab 9  was used for phase analysis of the sintered specimens. The instrument parameters were set as follows: The tube voltage was 40 kV, the current was 150 mA, Cu-k2 active specimens before and after sintering under different preforming pressures, and In order to observe the microstructure changes of the experimental specimen before and after sintering under different preforming pressure and the pressure holding time, the microstructure of the specimens before and after sintering was analyzed by means of scanning electron microscope (SEM). The SEM of 2 particles, not only the interface between TiH2 particles and Al particles and between Al and Al particles increases gradually, but also there are voids at the interface boundary. For each preformed pressure specimen, it has the same particle movement mode under external force loading, as shown in Unsintered: with the increase in preforming pressure, the deformation of particles gradually changed from the initial point contact to the surface contact, the contact area increases gradually, the interface between particles increases gradually, the sliding and deformation of grain boundary between particles decrease gradually, and the shape and orientation of voids are very irregular. Al particles change from spherical to flat with irregular orientation. From large irregular space to small irregular space. Because of the irregularity of TiHIt can be seen from Sintered: It can be seen from It can be seen from Combined with In order to facilitate the transformation between the engineering stress and strain and the real stress and strain, it can be considered that all experimental specimens always keep the cylindrical shape and volume unchanged during the compression process, as shown in 0, l0 is the initial height of the specimen, l is the instantaneous height of the specimen; F is the instantaneous loading stress measured by the universal material testing machine, S is the instantaneous area of the specimen, d is the instantaneous diameter of the specimen, The formula is as follows:eng is the engineering stress and εeng is the engineering strain. The following formulas of true strain (εtrue) and true stress (σtrue) can be obtained by combining with the above Equation (7).The original diameter of the specimen is d2 specimens were prepared under different preforming pressures.As shown in It can be seen from Combined with the above Equation (8), the visualization curve of the real stress–strain data of each group of experimental specimens 1, 2, 3, 4, 5 and 6 is given in 2 active material is a kind of typical strong and tough aluminum polytetrafluoroethyl composite, and its stress–strain curve can be divided into three stages: elastic deformation stage, strain hardening stage, failure stage of strain softening fracture [It can be seen from fracture . Its spefracture  to bear 2 active materials under different preforming pressures. The physical meaning of material toughness in the table indicates the energy absorbed per unit volume of specimen before fracture failure. The better the toughness, the higher the energy absorbed by the specimen, and the stronger the fracture resistance of the specimen. The visualized curves of real stress–strain data of active materials are greatly influenced by preforming pressure as a whole. It can be seen from 3 when the preforming pressure is 150 MPa, which is 2.8% higher than that of the specimen with the preforming pressure of 240 MPa, but its strength is slightly lower. Based on the above analysis, the material has high strength and strong fracture resistance when the preforming pressure is 240 MPa and the pressure holding time is 100 s.According to the formula of experimental mass loss rate, the strength of specimen with different preforming pressure and the pressure holding time can be characterized. The original mass M of the sample is 1.956 g and the residue mass m g. the formula of the experimental mass loss rate η  is as foAccording to the measured residual mass m, the mass loss rate of the specimen 1, 2, 3, 4, 5 and 6 can be obtained by using Equation (9), which is listed in According to 2 active material are the best when the preforming pressure is 240 MPa and the pressure holding time is 100 s. On this basis, the pressure holding time of 50 s, 150 s and 200 s was set, and the quasi-static compression experiment was carried out on the active material specimen. The specific active material parameters are shown in According to 2 specimen is 2.430 g/cm3 and 97.590% of the maximum theoretical density (TMD) when the pressure holding time is 50 s and the preforming pressure is 240 MPa.It can be seen from 2 specimens under different holding time are shown in Combined with the above Equation (8), the real stress–strain data visualization curves of Al-rich Al/PTFE/TiH3, respectively. In the deformation of the specimens with different the pressure holding times, the deformation of the specimen 4# is the smallest and the failure is the fastest. The density, compressive strength and toughness of the specimen 4# are the lowest, which are 2.397 g/cm3, 69.415 MPa and 81.700 MJ/m3, respectively. It may be that the density uniformity of specimen 4# is the worst during the pressing process. It is believed that the property of the material is decreased due to the fact that the PTFE matrix is not tightly bonded with the particles and the PTFE matrix is not completely coated with particles. The material with high strength and good toughness can be used in the warhead damage element to enhance the penetration effect on the target. According to According to Equation (9), the mass loss rate of specimens 1#, 2#, 3# and 4# can be obtained, as shown in It can be seen from 2 active specimen 5  photographed using a high-speed camera. The quasi-static compression process of all the specimens was recorded with a high-speed camera at a speed of 50 fps. 2 and open crack. By observing residues 1–6 and 1#–4#, it is found that there is no accumulated carbon black at each opening crack, as shown in 2 content of 10% did not react under the excess of Al, but it reacted at zero oxygen balance [2 is the key factor affecting the reaction of Al-rich Al/PTFE/TiH2 active materials under static loading. In order to further verify whether the Al-rich Al/PTFE/TiH2 specimens reacts, the phase analysis (XRD) of the residue after compression experiment is carried out, as shown in It can be seen from  balance . Therefo2 in the XRD patterns, but no diffraction peaks of other substances. It shows that the composition of Al-rich Al/PTFE/TiH2 residues is the same and no reaction occurs. Thus, it is proved that Al-rich Al/PTFE/TiH2 specimens do not react under static compression. Furthermore, excess Al may be the key factor affecting the non-reaction of Al-rich Al/PTFE/TiH2 under static loading. Therefore, the reactivity of Al-rich Al/PTFE/TiH2 specimens with TiH2 the content of 10% under quasi-static compression is not significantly affected by the compression parameters. It can be found from 2 specimens under molding parameters were studied by means of universal material testing machine and scanning electron microscope. X-ray diffractometer was used to analyze the phase of the residue after quasi-static compression experiment. The conclusions can be drawn as follows:Unsintered: with the increase in preforming pressure, the particles deform from point contact to surface contact, the contact area increases gradually, and the shape and orientation of voids are very irregular. When the preforming pressure is less than 150 MPa, the porosity of the specimen increases slowly at first and then decreases. When the preforming pressure is greater than 150 MPa, the porosity of the specimen increases first and then decreases. When the pressure holding time is no more than 100 s, the porosity of the specimen decreases gradually. When the pressure holding time is more than 100 s, the porosity of the specimen increases first and then decreases.Sintering: when the preforming pressure is less than 100 MPa, the porosity of the specimen decreases gradually. When the preforming pressure is greater than 100 MPa, the porosity of the specimen first increases and then decreases. With the increase in the pressure holding time, the porosity first increases and then decreases. For each preforming pressure specimen, compared with that before sintering, the porosity after sintering either decreases or increases. For each the pressure holding time specimen, the porosity increases after sintering compared with that before sintering. The microstructure of PTFE crystal in the specimen is mainly planar PTFE crystal. The size and number of planar PTFE crystals are significantly affected by the molding parameters. When the preforming pressure is less than 100 MPa, the planar PTFE crystals are small and less, the aggregates between PTFE matrix and particles are large and small, most particles are exposed, and the interfacial strength between particles is the lowest, which results in the worst mechanical properties of the specimens. When the preforming pressure is greater than 100 MPa and does not contain 240 MPa, the planar PTFE crystals are smaller and more, the voids are larger and more, and the interfacial strength between particles is higher, which results in better mechanical properties of the specimens. When the pressure holding time is 100 s, the planar PTFE crystals are large and more, and the interfacial strength between particles is the highest, which resulting in the best mechanical properties of the specimen.2 specimens first increase and then decrease. When the preforming pressure is lower than 240 MPa, the density of Al-rich Al/PTFE/TiH2 increases rapidly at first and then increases slowly. When the preforming pressure is not less than 240 MPa, the density of Al-rich Al/PTFE/TiH2 specimen is almost unchanged. When the preforming pressure is 240 MPa, the compressive strength of the specimen reaches the maximum, and its failure strain and toughness are better. When the preforming pressure is not less than 150 MPa, the maximum true strain of the specimen is more than 2, which indicates that the specimen with preforming pressure above 150 MPa has good ductility. Based on the above knowledge, the specimen with the preforming pressure of 240 MPa has the highest strength, better failure strain, better toughness and good ductility.With the increase in preforming pressures , the compressive strength, failure strain and toughness of Al-rich Al/PTFE/TiH2 specimens decreased, and their compressive strength and toughness increased first and then decreased. When the pressure holding time is 100 s, the density and failure strain of specimen are at moderate level, and its strength and toughness are the largest. The maximum true strain of specimens with different the pressure holding time is more than two, which indicates that the specimens with different the pressure holding time have good ductility. Based on the above knowledge, the overall mechanical properties of the specimen with the pressure holding time of 100 s are the best.With the increase in the pressure holding time , the density and failure strain of Al-rich Al/PTFE/TiH2 residues, and only diffraction peaks of Al, PTFE and TiH2 exist. It shows that Al-rich Al/PTFE/TiH2 specimens with TiH2 the content of 10% has no reaction occurred under quasi-static compression. It can be inferred that excess Al may be the key factor affecting the non-reaction of Al-rich Al/PTFE/TiH2 active materials under static loading. Therefore, the reactivity of Al-rich Al/PTFE/TiH2 specimens with TiH2 the content of 10% under quasi-static compression is not significantly affected by molding parameters.After quasi-static compression experiment, there are no diffraction peaks of other substances in the XRD patterns of all Al-rich Al/PTFE/TiHIn this paper, the mechanical properties and micromorphology of Al-rich Al/PTFE/TiH


The aim of this paper is to explore the mechanism of the change in oestrogenic activity of PCBs molecules before and after modification by designing new PCBs derivatives in combination with molecular docking techniques through the constructed model of oestrogenic activity of PCBs molecules. We found that the weakened hydrophobic interaction between the hydrophobic amino acid residues and hydrophobic substituents at the binding site of PCB derivatives and human oestrogen receptor alpha (hERα) was the main reason for the weakened binding force and reduced anti-oestrogenic activity. It was consistent with the information that the hydrophobic field displayed by the 3D contour maps in the constructed oestrogen activity CoMSIA model was one of the main influencing force fields. The hydrophobic interaction between PCB derivatives and oestrogen-active receptors was negatively correlated with the average distance between hydrophobic substituents and hydrophobic amino acid residues at the hERα-binding site, and positively correlated with the number of hydrophobic amino acid residues. In other words, the smaller the average distance between the hydrophobic amino acid residues at the binding sites between the two and the more the number of them, and the stronger the oestrogen activity expression degree of PCBS derivative molecules. Therefore, hydrophobic interactions between PCB derivatives and the oestrogen receptor can be reduced by altering the microenvironmental conditions in humans. This reduces the ability of PCB derivatives to bind to the oestrogen receptor and can effectively modulate the risk of residual PCB derivatives to produce oestrogenic activity in humans. Polychlorinated biphenyls (PCBs) are a type of persistent organic pollutants (POPs) ,2,3 and,1) and estradiol , whereas synthetic oestrogens are non-steroidal hormones. They play a vital role in the growth, development, and reproduction of the human body. Oestrogen can enter cells via transmembrane transport by binding to the corresponding receptors and activate the expression of several genes [Oestrogens are classified into two types: natural and synthetic. Natural oestrogens include steroids, such as estrone  method is widely used to study the relationship between the structure and activity data of molecules themselves ,20. Chu 20 of 11 PCBs measured by the in vitro dual luciferase reporter gene experiment as the 3D-QSAR modelling data. To facilitate subsequent QSAR analysis, the anti-oestrogen activity index of PCBs was calculated as the negative logarithm of the pREC20 value.Zhang et al.  used the20 values, PCB-44 with the largest pREC20 was selected as the template molecule. Among the data values of 11 PCBs with known pREC20 values, PCB-44 with the largest pREC20 was selected as the template molecule, seven kinds of PCBs were randomly selected as the training set and four kinds of PCBs were selected as the test set to build a 3D-QSAR model with a 2:1 ratio (ratio of the number of molecules in the training and test sets), and used the Minimize molecular program under SYBYL-X 2.0 to optimize the molecular mechanics of each molecule. The most stable conformation of the molecules was chosen . The Tripos force field combined with the minimize module was selected for energy optimization of each molecule to obtain the dominant stable conformation in the lowest energy state combined with the energy gradient method and load MMFF94 charge [The Surflex-Dock module in SYBYL-X2.0  was used to determine the difference in the effect of docking conformation of PCB molecules with human oestrogen receptor α (hERα) before and after modification. The target molecule PCB-44 and its 7 types of derivative molecules with low anti-oestrogenic activity were docked to each of the five hERα , indicating that the model had a reliable predictive capability [2 was 0.991 (>0.8), indicating that the model’s predictive ability was good. The standard deviation SEE was 0.146 and the F value was 114.424, indicating that the two models have a good fit and predictability [2pred was 0.902, SEP was 0.206, and Q2ext was 0.904, indicating that the model has a strong external predictive ability and robustness [20, indicating that the pREC20 values of the PCB homologues were affected by the spatial effect, electrical distribution, and hydrophobic interaction between the groups. Electrical distribution and hydrophobic field were the main influencing factors, whereas the hydrogen bond donor–acceptor field had no effect on them.The evaluation parameters of the CoMSIA model and the efficiency of the effect of the molecular force field are summarized in pability . In additability . The extbustness . In the 20 value was selected as the target molecule. 20 value of the PCB; namely, the activity of the PCB molecule was enhanced. 20 value of the PCBs. 20 value of PCBs. The t steric field, electrostatic field, and hydrophobic field, shown in the 3D contour map surround the PCB substitution site, which provided a reference for the selection of low oestrogen activity substitution sites. A comprehensive analysis of the contribution efficiency of each force field in the three-dimensional equipotential diagram of the CoMSIA model to the molecular structure revealed that the three-dimensional field, electrostatic field, and hydrophobic field mainly affect the three substitution sites of Cl3, Cl5, and Cl5′. In other words, introducing small, positively charged or hydrophilic groups at the positions of Cl3 and Cl5′, and introducing large, negatively charged or hydrophobic groups at the position of Cl5 all contributed to the reduction of the pREC20 value of the target molecule. Therefore, Cl3 and Cl5′ were selected as the sites for introducing the substitution groups and 8 types of the most common electronegative groups other than Cl 2, -C(CH3)3) and 4 common hydrophilic groups  were used as modification groups to produce 50 new PCB-44 molecular derivatives by single and double substitution reactions.The 3D contour map of the CoMSIA model is shown in 20 values of 50 types of novel PCB-44 molecular derivatives were predicted using the 3D-QSAR model and the predicted results are summarized in 20 values of PCB-44 derivative molecules ranged from 0.6% to 38.7%; that is, the anti-oestrogenic activity of PCB-44 derivatives was significantly reduced. Among them, the more prominent ones were 5′-tert-butyl-PCB-44 and 3-methyl-5′-methyl-PCB-44, and, compared to PCB-44, their anti-oestrogenic activities were reduced by 38.7% and 37.1%, respectively. This showed that the PCB-44 derivatives designed based on the CoMSIA model had a significant effect on the reduction in anti-oestrogenic activities. We further proved that the change in the structures of PCB molecules can effectively reduce their anti-oestrogenic activity. In addition, the properties of POPs  of PCB-ntration . After sIn this study, we used the Surflex-Dock module in SYBYL-X2.0 to bind the 7 types of novel and target PCB molecules exhibiting low anti-oestrogenic activity to each of the 5 types of hERα , respectively. The results are given in As summarized in In summary, the fewer the hydrophobic residues surrounding the new PCB molecules, the less hydrophobic the interaction between them, and the more loosely bound they are, the lower anti-oestrogenic activity is expressed by the PCB molecules. The lesser the average distance between the hydrophobic substituents of PCB derivatives and the hydrophobic amino acid residues of the oestrogen receptor-binding site, the stronger the hydrophobic interaction between the two. That is, the stronger the binding force between the PCB molecule and the oestrogen receptor, the stronger the anti-oestrogenic activity of the PCB molecule, thus further explaining the differential anti-oestrogenic activity of the PCB-44 derivative molecule when bound to the oestrogen receptor.4) Taguchi experiment [We further screened the conditions of ingested substances in vivo conditions of the human body that could be adjusted to reduce the effect of PCBs before and after modification to bind to oestrogen receptors in the human body in order to prevent the expression of oestrogen activity. Thus, we created a partial factorial design of 7 PCB derivatives with reduced anti-oestrogenic activity using molecular dynamics assisted by the L9(3periment  to calcuperiment  showed tperiment  (pumpkinperiment  (apple, periment  (grape, periment  (the 4 fPrevious studies  have shoWe used the 3D-QSAR model combined with molecular docking technology to investigate in detail the mechanism of differential expression of anti-oestrogenic activity of PCB molecules before and after modification, which was significantly and positively correlated with the hydrophobic interaction between PCB molecules and oestrogenic receptors. The main factor affecting the hydrophobic interaction between PCBs before and after modification and the oestrogen receptor was the number of hydrophobic amino acid residues and the average distance between them as well as the hydrophobic substituent; the intake of xylitol, mannitol, tannins, and anthocyanin in certain proportions significantly attenuated the hydrophobic effect of the microenvironment in which the amino acid residues of PCB molecules bound to oestrogenic receptors by using molecular dynamics simulations assisted by Taguchi’s experimental design. That is, adjusting the human microenvironment can indirectly affect the expression of estrogen activity of PCBs molecules exposed to the human environment, and alleviate the risk of estrogen activity of residual PCBs molecules in the human body. In this paper, the main factors affecting the estrogenic activity of PCBs molecules and the designed adjustment scheme of human intake microenvironment are determined, which provide a theoretical basis and new ideas for the risk of estrogenic activity produced by PCBs molecules.