Pseudomonas aeruginosa has become increasingly challenging, resulting in a worse morbidity and mortality. Airway remodeling is a common phenomenon in this process, to which epithelial-mesenchymal transition (EMT) may contribute as an important promoter. Previous studies showed that epithelium-specific integrin αvβ6–mediated EMT was involved in pulmonary fibrosis via transforming growth factor-β1 (TGF-β1) signaling, but whether integrin αvβ6 plays a role in the P. aeruginosa–associated airway remodeling remains unknown. BEAS-2B cells were incubated with lipopolysaccharide (LPS) from P. aeruginosa in the presence or the absence of integrin αvβ6–blocking antibodies. Morphologic changes were observed by an inverted microscopy. The EMT markers were detected using Western blotting and immunofluorescence. The activation of TGF-β1-Smad2/3 signaling pathway was assessed. Furthermore, matrix metalloproteinase (MMP)-2 and -9 in the medium were measured using ELISA. P. aeruginosa’s LPS decreased the expression of the epithelial marker E-cadherin and promoted the mesenchymal markers, vimentin and α-smooth muscle actin in BEAS-2B cells. The expression of integrin αvβ6 was significantly increased during EMT process. Blocking integrin αvβ6 could attenuate P. aeruginosa’s LPS-induced EMT markers’ expression via TGF-β1-Smad2/3 signaling pathway. Furthermore, blocking integrin αvβ6 could prevent morphologic changes and oversecretion of MMP-2 and -9. Integrin αvβ6 mediates epithelial-mesenchymal transition in human bronchial epithelial cells induced by lipopolysaccharides of P. aeruginosa via TGF-β1-Smad2/3 signaling pathway and might be a promising therapeutic target for P. aeruginosa–associated airway remodeling.Lower respiratory tract infection due to  Pseudomonas aeruginosa is one of the most frequent opportunistic pathogens, responsible for serious lower respiratory tract infection in patients with cystic fibrosis  has been identified as an important source of fibroblasts that could contribute to the remodeling of the airways. EMT is a reversible process by which epithelial cells change the phenotype and function into that of fibroblast-like, mesenchymal ones, characterized by loss of epithelial markers, acquisition of mesenchymal markers, and matrix metalloproteinase production  is a pleiotropic growth factor which has been considered a key inducer of fibrosis and has a central role in regulating EMT process by mediating Smad-dependent and Smad-independent signaling pathways . BEAS-2B cells were pretreated with Integrin αvβ6–blocking antibody  or TGF-β1-Smad2/3 signaling inhibitor, SB431542 , for 2 h prior to incubation with 2 μg/mL P. aeruginosa’s LPS  at 37 °C in a humidified 5% COv/v) NP-40, 0.1% (w/v) SDS) containing 1% (v/v) phenylmethylsulfonyl fluoride (SolarBio), 0.3% (v/v) protease inhibitor (Sigma Aldrich), and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma) and then clarified by centrifugation. The supernatant was collected and then separated on an SDS-PAGE gel (10% (v/v) polyacrylamide), transferred onto a PVDF membrane. Nonspecific binding was blocked in Tris-buffered saline with Tween 20 (TBS-T) with 8% (w/v) milk for 2 h. After incubation with primary antibodies against β-actin (Abmart), E-cadherin , vimentin , α-smooth muscle actin , Smad2/3 (Abcam), and p-Smad2/3 (Abcam) for overnight at 4 °C, the membranes were washed with TBS-T for several times and incubated in HRP-linked secondary antibodies (Abmart) for 2 h at room temperature (RT). After repeated washing with TBS-T, the immunoreactive proteins were visualized using enhanced chemiluminescence (Millipore) according to the manufacturer’s instructions and quantified using density analysis, normalized against β-actin and expressed as the fold change compared with the control.Cells were lysed in RIPA buffer  three times and then blocked in 3% bovine serum album (BSA) for 60 min at RT. The cells were incubated with primary antibodies against human E-Cad and Vi (1:100 dilution in PBS with 1% BSA) for 2 h at RT. After several washes with PBS, the slides were incubated with Alexa Fluor 488–conjugated anti-rabbit IgG  for 60 min at RT. After several washes for 15 min with PBS, the slides were incubated with Hoechst 33258 (10 μg/mL) for 10 min at RT. Finally, the slides were washed again, mounting reagent was added, and images were captured using a fluorescence microscope.To determine the secretion of active TGF-β1, metalloproteinase (MMP)-2, and MMP-9, ELISA procedure was performed according to the manufacturer’s instructions (Beijing Rui’erxinde Technology).p < 0.05 was considered significant.Data were expressed as mean ± SEM. Significance of the results were analyzed by performing one-way ANOVA, except time-dependent changes of the expression of EMT markers and integrin αvβ6 in BEAS-2B cells which were analyzed with repeated measures ANOVA test. Tukey’s post hoc tests were performed for multiple comparisons. P. aeruginosa’s LPS could induce EMT in BEAS-2B cells. After incubation with P. aeruginosa’s LPS (2 μg/mL), the expression of the epithelial marker, E-Cad, and mesenchymal markers, Vi and α-SMA, in BEAS-2B cells were detected using Western blotting at different time points , a receptor for LPS which could enhance TGF-β signaling pathway, has been reported to play a key role in the process of organ fibrogenesis and LPS-induced EMT process(Guillot et al. P. aeruginosa’s LPS. Our results showed that P. aeruginosa’s LPS induced BEAS-2B cells to transdifferentiate into a fibroblast-like, spindle-shaped phenotype instead of the original cobblestone-like morphology, which could be alleviated by integrin αvβ6–blocking antibody 10D5. The finding is consistent with a previous study that integrin αvβ6 can regulate morphologic changes during EMT process (Ramos et al. In this study, we also examined whether integrin αvβ6 could regulate morphologic changes and MMP secretion induced by P. aeruginosa’s LPS, which were significantly attenuated by blocking integrin αvβ6. These data indicated that integrin αvβ6 could regulate MMP-2 and -9 secretion accompanied with EMT in BEAS-2B cells, which was consistent with the studies from Ahmed et al. (MMP-2 and -9 have been demonstrated to be important contributors to fibrosis process by activating TGF-β1 and degrading basement membranes (Corbel et al. d et al.  and Thomd et al. .P. aeruginosa’s LPS is sufficient to induce EMT in human bronchial epithelial cells which in turn could contribute to airway fibrotic remodeling. Furthermore, our results obtained here suggest that P. aeruginosa’s LPS-induced EMT in BEAS-2B cells could be regulated by integrin αvβ6–mediated TGF-β1-Smad2/3 signaling activation, and blocking αvβ6 could abrogate EMT changes of BEAS-2B cells, including expression of EMT markers, cell morphology, and secretion of MMP-2 and -9. Thus, integrin αvβ6 might be a safe and effective therapeutic target for P. aeruginosa–associated airway remodeling (Fig. In summary, this study demonstrated that


TM SiC/[Si-B-C] and 2D woven Hi-NicalonTM SiC/[SiC-B4C] ceramic-matrix composites (CMCs) at 600 and 1200 °C was investigated. The cycle-dependent damage parameters of internal friction, dissipated energy, Kachanov’s damage parameter, and broken fiber fraction were obtained to describe damage development in self-healing CMCs. The relationships between cycle-dependent damage parameters and multiple fatigue damage mechanisms were established. The experimental fatigue damage development of self-healing Hi-NicalonTM SiC/[Si-B-C] and Hi-NicalonTM SiC/[SiC-B4C] composites was predicted for different temperatures, peak stresses, and loading frequencies. The cycle-dependent damage evolution of self-healing Hi-NicalonTM SiC/[Si-B-C] and Hi-NicalonTM SiC/[SiC-B4C] composites depends on temperature, testing environment, peak stress, and loading frequency. For the Hi-NicalonTM SiC/[Si-B-C] composite, temperature is a governing parameter for the fatigue process. At an elevated temperature of 600 °C in an air atmosphere, the internal frictional parameter of Hi-NicalonTM SiC/[Si-B-C] composite decreases first and then increases with applied cycle number; however, at an elevated temperature of 1200 °C in an air atmosphere, the internal frictional parameter of Hi-NicalonTM SiC/[Si-B-C] composite decreases with applied cycle number, and the interface shear stress at 1200 °C is much lower than that at 600 °C. For Hi-NicalonTM SiC/[SiC-B4C] composite at 1200 °C, loading frequency is a governing parameter for the fatigue process. The degradation rate of interface shear stress is much higher at the loading frequency of 0.1 Hz than that at the loading frequency of 1 Hz.Cycle-dependent damage evolution in self-healing, 2.5D woven Hi-Nicalon The lifetime duration is about t = 10–20 h at T = 873 K and less than t = 1 h at T = 1123 K under σmax = 120 MPa. For the self-healing Hi-NicalonTM SiC/SiC composite, a duration of t = 1000 h without failure is reached at σmax = 170 MPa, and a duration higher than t = 100 h at σmax = 200 MPa and T = 873 K is reached [TM SiC/[SiC-B4C] composite, at 1200 °C, there is little influence on the fatigue performance at f = 1.0 Hz, but there is a noticeably degraded fatigue lifetime at f = 0.1 Hz with the presence of steam [TM SiC/[Si-B-C] at T = 873 K; and the fiber creep controls the fatigue lifetime of self-healing SiC/[Si-B-C] at T = 1200 °C [TM SiC/[Si-B-C] composite involves an initial decrease of the effective modulus to a minimum value, followed by a stiffening, and the time-to-the minimum modulus is in inverse proportion to the loading frequency [TM SiC/[Si-B-C] composite at elevated temperature can be monitored using acoustic emission (AE)  and 2D woven Hi-NicalonTM SiC/[SiC-B4C] composites at T = 600 °C and 1200 °C using damage evolution models and parameters. The cycle-dependent damage parameters of internal friction, dissipated energy, Kachanov’s damage parameter, and broken fiber fraction were obtained to analyze damage development in self-healing CMCs. The relationships between cycle-dependent damage parameters and multiple fatigue damage mechanisms are established. The experimental fatigue damage evolution of each of the composites—self-healing Hi-NicalonTM SiC/[Si-B-C] and Hi-NicalonTM SiC/[SiC-B4C]—is predicted. The effects of fatigue peak stress, testing environment, and loading frequencies on the evolution of internal damage and final fracture are analyzed.The objective of this paper is to analyze cycle-dependent damage development in self-healing 2.5D woven Hi-NicalonTM fibers  with an interphase of pyrolytic carbon (PyC) reinforced multilayered matrix [Si-B-C] were provided by SNECMA Propulsion Solide, Le Haillan, France. The self-healing Hi-NicalonTM SiC/[Si-B-C] composite was fabricated using chemical vapor infiltration (CVI). The experimental results were performed and obtained by Penas [TM SiC/[Si-B-C] composite at 600 °C and 1200 °C in an air atmosphere are shown in The Hi-Nicalonby Penas . The detmin = −50 MPa; and for tension–tension fatigue tests, the minimum stress is set to be σmin = 0 MPa, and the fatigue limit was defined to be N = 1,000,000.The fatigue specimen was dog bone-shaped, and the dimensions were 200 mm total length, 5 mm thickness, and 16 mm width in the gage section. The fatigue experiments at elevated temperature were conducted in an INSTRON Model 8502  and an AET furnace system . The fatigue tests were on a sinusoidal wave form and the loading frequency was f = 0.25 Hz. For tension–compression fatigue tests, the minimum stress is always equal to σTM SiC/[SiC-B4C] composite was reinforced with Hi-NicalonTM fibers with the PyC interphase (about 0.4 μm thick) and boron carbide overlay (about 1.0 μm thick), and was processed via CVI by Hyper-Therm High-Temperature Composites, Inc. . The experimental results were obtained by Ruggles-Wrenn et al.  and 2D woven Hi-NicalonTM SiC/[SiC-B4C]—when subjected to cyclic fatigue loading, was obtained.The monotonic tensile and cycle-dependent damage evolutions of self-healing 2.5D woven Hi-NicalonTM SiC/[Si-B-C] composite at T = 600 °C in an air atmosphere is shown in TM SiC/[Si-B-C] composite fractures at σUTS = 341 MPa with the failure strain of εf = 0.64%. The tensile curve exhibits obvious nonlinearity, and can be divided into three zones, including: (1) the linear elastic zone with an elastic modulus of Ec = 195 ± 20 GPa; (2) the nonlinear zone due to multiple matrix cracking; and (3) the second linear zone after saturation of matrix cracking up to final fracture, with an elastic modulus of Ec = 23 ± 1 GPa, which is half of theoretical value EfVfl (43 GPa) when the load is supported only by the longitudinal fiber.The monotonic tensile curve of 2.5D woven self-healing Hi-Nicalone) versus cycle number curves of 2.5D woven self-healing Hi-NicalonTM SiC/[Si-B-C] composite under σmin = −50/σmax = 300 MPa and σmin = 0/σmax = 200 MPa at T = 600 °C in an air atmosphere are shown in e) decreases first, followed by a short stabilization, and increases again before reaching a plateau; and finally, there is a sharp increase when the composite approaches failure. During initial stage of cyclic fatigue loading, matrix cracking and interface debonding occur when the fatigue peak stress is higher than the first matrix cracking stress. Under repeated unloading and reloading, the sliding between the fiber and the matrix leads to the interface wear and oxidation, which decreases the interface shear stress  at 600 °C in an air atmosphere are shown in max = 200 and 300 MPa, the internal damage parameter (∆W(N)/We(N)) first decreases with the interface shear stress, mainly due to the interface wear and oxidation, and then increases with the interface shear stress, mainly due to the fiber broken, corresponding to the interface slip Case 4 in max = 300 MPa, the broken fiber fraction (P(N)) at higher interface shear stress is much higher than that under σmax = 200 MPa, mainly due to higher peak stress, as shown in max = 200 MPa, as shown in max = 300 MPa is also higher than that under σmax = 200 MPa, mainly due to the scatter of interface shear stress or compressive stress of σmin = −50 MPa acting on the composite.The experimental and predicted cycle-dependent internal friction parameter (∆W(N)/Wmax = 200 MPa, the cycle-dependent damage parameter (∆W/We) decreases first, i.e., from ∆W/We = 0.054 at τi = 15.7 MPa to ∆W/We = 0.034 at τi = 8.0 MPa, and then increases from ∆W/We = 0.034 at τi = 8.0 MPa to ∆W/We = 0.062 at τi = 6.0 MPa. The cycle-dependent broken fiber fraction (P) increases from P = 0.004 at τi = 15.7 MPa to P = 0.24 at τi = 6.0 MPa. The cycle-dependent Kachanov’s damage parameter (D) increases from D = 0 at N = 1 to D = 0.21 at N = 89,459. The cycle-dependent interface shear stress (τi) decreases from τi = 15.7 MPa at N = 1 to τi = 6.0 MPa at N = 33,788.Under σmax = 300 MPa, the cycle-dependent damage parameter (∆W/We) decreases first, i.e., from ∆W/We = 0.067 at τi = 19.5 MPa to ∆W/We = 0.058 at τi = 14.7 MPa; and then increases from ∆W/We = 0.058 at τi = 14.7 MPa to ∆W/We = 0.091 at τi = 10.5 MPa. The cycle-dependent broken fiber fraction (P) increases from P = 0.029 at τi = 19.5 MPa to P = 0.347 at τi = 10.5 MPa. The cycle-dependent Kachanov’s damage parameter (D) increases from D = 0 at N = 1 to D = 0.265 at N = 23,666. The interface shear stress (τi) decreases from τi = 19.5 MPa at N = 1 to τi = 10.5 MPa at N = 19,812.Under the peak stress of σTM SiC/[Si-B-C] composite at T = 1200 °C in an air atmosphere is shown in UTS = 354 MPa with εf = 0.699%. The tensile curve can also be divided into three zones, including: (1) the initial linear elastic zone; (2) the non-linear zone; and (3) the second linear region with fiber broken. The average fracture strength and failure strain of 2.5D woven Hi-NicalonTM SiC/[Si-B-C] composite are slightly lower at T = 1200 °C; i.e., σUTS = 320 MPa and ɛf = 0.62%, against σUTS = 332 MPa and ɛf = 0.658% at T = 600 °C.The monotonic tensile curve of 2.5D woven self-healing Hi-Nicalone) versus cycle number curves of 2.5D woven self-healing Hi-NicalonTM SiC/[Si-B-C] at T = 1200 °C in an air atmosphere are shown in e) decreases continuously, and finally, there is a sharp increase when the composite approaches failure. The internal friction decreases as the interface wear reduces the interface shear stress. The sudden increase of internal friction at the end of the test corresponds to the fiber broken.The experimental cycle-dependent internal friction parameter (∆W/We(N)) and the broken fiber fraction (P(N)) versus the interface shear stress curves; and the cycle-dependent Kachanov’s damage parameter (D(N)) and the interface shear stress (τi(N)) versus cycle number curves of 2.5D woven self-healing Hi-NicalonTM SiC/[Si-B-C] composite at 1200 °C in an air atmosphere are shown in max = 170 and 200 MPa, the internal damage parameter (∆W/We) decreases with the interface shear stress, corresponding to the interface slip case (Case 4) in max = 200 MPa, the broken fiber fraction (P) is higher than that under σmax = 170 MPa at the same interface shear stress; and the Kachanov’s damage parameter (D) is also higher than that under σmax = 170 MPa at the same cycle number. However, the value of the interface shear stress under σmax = 200 MPa is close to that under σmax = 170 MPa.The experimental and predicted cycle-dependent internal friction parameter (∆W(N)/Wmax = 170 MPa, the cycle-dependent internal friction parameter (∆W/We) increases to the peak value first and then decreases; i.e., from ∆W/We = 0.2 at τi = 150 MPa to ∆W/We = 0.42 at τi = 39.5 MPa, and then to ∆W/We = 0.04 at τi = 1.25 MPa. The broken fiber fraction (P) increases from P = 0.0007 at τi = 150 MPa to P = 0.12 at τi = 1.2 MPa. The Kachanov’s damage parameter (D) increases from D = 0 at N = 1 to D = 0.068 at N = 13,202. The interface shear stress (τi) decreases from τi = 5.5 MPa at N = 1 to τi = 1.7 MPa at N = 32334.Under σmax = 200 MPa, the cycle-dependent internal friction parameter (∆W/We) increases to the peak value first and then decreases; i.e., from ∆W/We = 0.161 at τi = 150 MPa to ∆W/We = 0.42 at τi = 26.4 MPa, and then to ∆W/We = 0.05 at τi = 1.37 MPa. The broken fiber fraction (P) increases from P = 0.0008 at τi = 150 MPa to P = 0.14 at τi = 1.3 MPa. The Kachanov’s damage parameter (D) increases from D = 0 at N = 1 to D = 0.2 at N = 60530. The interface shear stress (τi) decreases from τi = 5.1 MPa at N = 1 to τi = 2 MPa at N = 66794.Under σTM SiC/[SiC-B4C] composite at T = 1200 °C in an air atmosphere is shown in UTS = 345 MPa with εf = 0.79%. The experimental cycle-dependent dissipated energy (ΔW(N)) versus cycle number curves of 2D woven self-healing Hi-NicalonTM SiC/[SiC-B4C] composite under σmax = 140 MPa at T = 1200 °C in an air atmosphere and in a steam atmosphere with f = 0.1 and 1 Hz are shown in The monotonic tensile curve of 2D woven self-healing Hi-Nicaloni(N)) and dissipated energy (ΔW(N)) versus cycle number curves of 2D woven self-healing Hi-NicalonTM SiC/[SiC-B4C] composite under σmax = 140 MPa and f = 0.1 and 1 Hz at T = 1200 °C in air and in steam atmospheres are shown in The experimental and predicted cycle-dependent dissipated energy (ΔW(N)) and broken fiber fraction (P(N)) versus the interface shear stress curves, and the cycle-dependent interface shear stress  increases from ΔW = 8.5 kPa at τi = 34 MPa to ΔW = 32.4 kPa at τi = 9.2 MPa; the broken fiber fraction (P) increases from P = 0.001 at τi = 150 MPa to P = 0.167 at τi = 1.57 MPa; the experimental interface shear stress (τi) decreases from τi = 34 MPa at N = 1000 to τi = 9.2 MPa at N = 60,000; and the experimental dissipated energy (ΔW) increases from ΔW = 8.4 kPa at N = 1000 to ΔW = 32.4 kPa at N = 60,000.At T = 1200 °C in air, under σmax = 140 MPa and f = 0.1 Hz, the experimental cycle-dependent dissipated energy (ΔW) increases from ΔW = 12.7 kPa at τi = 23 MPa to ΔW = 34.2 kPa at τi = 8.7 MPa; the broken fiber fraction (P) increases from P = 0.001 at τi = 150 MPa to P = 0.167 at τi = 1.57 MPa; the experimental interface shear stress (τi) decreases from τi = 23 MPa at N = 1000 to τi = 8.7 MPa at N = 30,000; and the experimental dissipated energy (ΔW) increases from ΔW = 12.7 kPa at N = 1000 to ΔW = 34.2 kPa at N = 30,000.Under σmax = 140 MPa and f = 1 Hz, the experimental cycle-dependent dissipated energy (ΔW) increases from ΔW = 8.5 kPa at τi = 34 MPa to ΔW = 18.1 kPa at τi = 16.3 MPa; the broken fiber fraction (P) increases from P = 0.001 at τi = 150 MPa to P = 0.167 at τi = 1.57 MPa; the experimental interface shear stress (τi) decreases from τi = 34 MPa at N = 1000 to τi = 16.3 MPa at N = 30,000; and the experimental dissipated energy (ΔW) increases from ΔW = 8.5 kPa at N = 1000 to ΔW = 18.1 kPa at N = 30,000.At T = 1200 °C in steam, under σmax = 140 MPa and f = 0.1 Hz, the experimental cycle-dependent dissipated energy (ΔW) increases with decreasing interface shear stress—i.e., from ΔW = 14.8 kPa at τi = 20 MPa to ΔW = 27 kPa at τi = 11 MPa; the broken fiber fraction (P) increases from P = 0.001 at τi = 150 MPa to P = 0.167 at τi = 1.57 MPa; the experimental interface shear stress (τi) decreases from τi = 20 MPa at N = 100 to τi = 11 MPa at N = 10000; and the experimental dissipated energy (ΔW) increases from ΔW = 14.8 kPa at N = 100 to ΔW = 27 kPa at N = 10000.At T = 1200 °C in steam, under σi(N)) and broken fiber fraction (P(N)) versus cycle number of 2.5D woven self-healing Hi-NicalonTM SiC/[Si-B-C] composite under different peak stresses at T = 600 °C and 1200 °C in an air atmosphere are shown in The evolution curves of cycle-dependent interface shear stress  decreases from τi = 15.7 MPa at N = 1 to τi = 6.0 MPa at N = 33788, and the broken fiber fraction (P) increases from P = 0.004 to P = 0.24; and under σmax = 300 MPa, the interface shear stress (τi) decreases from τi = 19.5 MPa at N = 1 to τi = 10.5 MPa at N = 19,812, and the broken fiber fraction (P) increases from P = 0.029 to P = 0.347. However, at T = 1200 °C and under σmax = 170 MPa, the interface shear stress (τi) decreases from τi = 5.5 MPa at N = 1 to τi = 1.7 MPa at N = 32,334, and the broken fiber fraction (P) increases from P = 0.0007 to P = 0.12; and under σmax = 200 MPa, the interface shear stress (τi) decreases from τi = 5.1 MPa at N = 1 to τi = 2 MPa at N = 66794, and the broken fiber fraction (P) increases from P = 0.0008 to P = 0.14.At T = 600 °C and σi(N)) and the broken fiber fraction (P(N)) versus cycle number of 2D woven self-healing Hi-NicalonTM SiC/[SiC-B4C] composite under σmax = 140 MPa at T = 1200 °C in air and in steam atmospheres with f = 0.1 and 1Hz are shown in 4C] composite, the loading frequency is the governing parameter for the fatigue process. Under σmax = 140 MPa, at the same cycle number, the interface shear stress (τi) decreases with decreasing loading frequency, and the interface shear stress in a steam atmosphere is lower than that in an air atmosphere; and the broken fiber fraction (P) increases with decreasing loading frequency, and the broken fiber fraction in a steam atmosphere is higher than that in an air atmosphere.The evolution curves of cycle-dependent interface shear stress (τi) decreases from τi = 34 MPa at N = 1000 to τi = 9.2 MPa at N = 60,000, and the broken fiber fraction (P) increases from P = 0.0048 to P = 0.018; when f = 0.1 Hz, the interface shear stress (τi) decreases from τi = 23 MPa at N = 1000 to τi = 8.7 MPa at N = 30,000, and the broken fiber fraction (P) increases from P = 0.007 to P = 0.02. At T = 1200 °C in a steam atmosphere and f = 1 Hz, the interface shear stress (τi) decreases from τi = 34 MPa at N = 1000 to τi = 16.3 MPa at N = 30,000, and the broken fiber fraction (P) increases from P = 0.0048 to P = 0.01; and when f = 0.1 Hz, the interface shear stress (τi) decreases from τi = 20 MPa at N = 100 to τi = 11 MPa at N = 10000, and the broken fiber fraction (P) increases from P = 0.008 to P = 0.015.At T = 1200 °C in an air atmosphere with f = 1 Hz, the interface shear stress , dissipated energy (∆W), Kachanov’s damage parameter (D), broken fiber fraction (P), and interface shear stress (τi) are used to describe fatigue damage evolution.In this paper, cycle-dependent damage evolution of self-healing 2.5D woven Hi-Nicalon(1)TM SiC/[Si-B-C] composite, temperature is a governing parameter for the fatigue process. At T = 600 °C in an air atmosphere, ∆W/We first decreases and then increases with cycle number; and at T = 1200 °C in an air atmosphere, ∆W/We decreases with cycle number. The degradation rate of the interface shear stress and broken fiber faction increases with peak stress.For 2.5D woven self-healing Hi-Nicalon(2)TM SiC/[SiC-B4C] composite at T = 1200 °C, loading frequency is a governing parameter for the fatigue process. ∆W increases with cycle number; under σmax = 140 MPa, at the same applied cycle number, the interface shear stress decreases with the loading frequency, and the interface shear stress in a steam atmosphere is lower than that in an air atmosphere, and the broken fiber fraction increases with decreasing loading frequency, and the broken fiber fraction in a steam atmosphere is higher than that in an air atmosphere.For 2D woven self-healing Hi-Nicalon


The chains pack with normal van der Waals contacts.The dihydroquinoxaline moiety in the title compound is not planar. In the crystal, C—H⋯O hydrogen bonds form helical chains about the crystallographic 2 16H14N2O, the dihydroquinoxaline moiety is not planar as there is a dihedral angle of 4.51 (5)° between the constituent rings. In the crystal, C—H⋯O hydrogen bonds form helical chains about the crystallographic 21 screw axis in the b-axis direction. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯H (51.7%), H⋯C/C⋯H (26%) and H⋯O/O⋯H (8.5%) interactions. The optimized structure calculated using density functional theory (DFT) at the B3LYP/6–311 G level is compared with the experimentally determined structure in the solid state. The calculated HOMO–LUMO energy gap is 3.8918 eV.In the title molecule, C In addition to the synthesis, we also report the molecular and crystal structure along with a Hirshfeld surface analysis and a density functional theory (DFT) computational study carried out at the B3LYP/6–311 G level.Nitrogen-based structures have attracted attention in recent years because of their interesting properties in structural and inorganic chemistry (Chkirate The molecular structure of (I)1 axes are formed by C9—H9B⋯O1 hydrogen bonds , Fig. 5c, have tips at de + di = 2.79 Å. The pair of scattered points of spikes in the fingerprint plot delineated into H⋯O/O⋯H, Fig. 5e (8.5%), have the tips at de + di = 2.26 Å. The C⋯C contacts, Fig. 5f (6.1%), have the tips at de + di = 3.45 Å. The H⋯N/N⋯H contacts, Fig. 5d, contribute 6% to the HS and appear as a pair of scattered points of spikes with the tips at de + di = 2.67 Å. The C⋯N/N⋯C contacts, Fig. 5g, contribute 1.5% to the HS, appearing as pair of scattered points of spikes with the tips at de + di = 3.30 Å. Finally, the C⋯O/O⋯C contacts, Fig. 5h, make only a 0.2% contribution to the HS and have a low-density distribution of points.In order to visualize the intermolecular interactions in the crystal of the title compound, a Hirshfeld surface (HS) analysis , hardness (η), ionization potential (I), dipole moment (μ), electron affinity (A), electrophilicity (ω) and softness (σ) are collated in Table 3ΔE = ELUMO - EHOMO] of the molecule is 3.8918 eV, and the frontier molecular orbital energies, EHOMO and ELUMO, are −6.1381 and −2.2463 eV, respectively.The optimized structure of (I)et al., 2016et al., 2018aet al., 2013et al., 2005aet al., 2018H-1,2,3-triazol-4-yl]methyl)-3-methyl; Abad et al., 2020H-1,2,3-triazol-4-yl)methyl]-3-methyl; Abad et al., 2018bet al., 2005bA search of the Cambridge Structural Database -one  in N,N-dimethylformamide (20 ml) were added bromoethane (0.48 ml), potassium carbonate K2CO3  and a catalytic quantity of tetra-n-butylammonium bromide. The reaction mixture was stirred at room temperature for 24 h. The solution was filtered and the solvent removed under reduced pressure. The residue thus obtained was separated by chromatography on a silica gel column using a hexane/ethyl acetate 9:1 mixture as eluent. The solid obtained was recrystallized from ethanol solution to afford colourless plates of the title compound (yield: 85%).To a solution of 3-phenylquinoxalin-2(1Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020015819/pk2652sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989020015819/pk2652Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020015819/pk2652Isup3.cmlSupporting information file. DOI: 2047850CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


C-Analogues of the canonical N-nucleosides have considerable importance in medicinal chemistry and are promising building blocks of xenobiotic nucleic acids (XNA) in synthetic biology. Although well established for synthesis of N-nucleosides, biocatalytic methods are lacking in C-nucleoside synthetic chemistry. Here, we identify pseudouridine monophosphate C-glycosidase for selective 5-β-C-glycosylation of uracil and derivatives thereof from pentose 5-phosphate  substrates. Substrate requirements of the enzymatic reaction are consistent with a Mannich-like addition between the pyrimidine nucleobase and the iminium intermediate of enzyme (Lys166) and open-chain pentose 5-phosphate. β-Elimination of the lysine and stereoselective ring closure give the product. We demonstrate phosphorylation-glycosylation cascade reactions for efficient, one-pot synthesis of C-nucleoside phosphates (yield: 33 – 94%) from unprotected sugar and nucleobase. We show incorporation of the enzymatically synthesized C-nucleotide triphosphates into nucleic acids by RNA polymerase. Collectively, these findings implement biocatalytic methodology for C-nucleotide synthesis which can facilitate XNA engineering for synthetic biology applications. C-nucleosides are analogues of the canonical N-nucleosides and, despite their synthetic value, biocatalysis has not targeted them yet. Here, the authors report a pseudouridine monophosphate C-glycosidase enzyme for selective 5-β-C-glycosylation of uracil and its derivatives from pentose 5- phosphate substrates. N-glycosidic) linkage. Nucleoside analogs replacing the canonical N- by a rare-to-nature C-glycosidic linkage resist the common routes of biological degradation by N-glycoside hydrolases and phosphorylases4. Since in vivo stability is crucial for compound efficacy and applicability in therapy, synthetic C-nucleosides have considerable importance in medicinal chemistry. Nucleoside analogs generally represent promising lead structures in drug design for antiviral, antibacterial and antitumor activity9. Synthetic C-nucleosides, such as BCX4430  and GS-6620 36. For example, the uniform substitution of U by Ψ can render synthetic mRNA non-immunogenic and was shown to enhance the translation efficiency32. Additionally, Ψ-modified mRNA can boost the gene-cutting efficiency in CRISPR-based gene editing38. Ψ; Fig.  occurs no U Fig. , positioC-nucleosides for their emerging applications in XNA engineering or in drug design, the target compounds must be made available through efficient and structurally precise synthetic methods. Chemical routes to C-nucleosides typically follow the synthetic rationale of (1) reconstructing the heteroaryl nucleobase on a C1′-functionalized sugar or (2) coupling the prepared heteroaryl with a suitable glycosyl reagent, such as a hydroxy group-protected glycal or pentonolactone43. However, despite much progress from research over decades, a purely chemical approach to defined C-nucleosides remains challenging to establish50. A synthesis that exploits the high stereo- and regiocontrol of enzymatic reactions for installing the C-glycosidic linkage would be highly desirable. Natural enzymes of C-glycoside biosynthesis, such as tRNA pseudouridine synthase52 and C-glycosynthase54 have complex substrate requirements, leading to perceived limitations on their applicability. Therefore, unlike N-nucleosides for which N-nucleoside phosphorylases present excellent tools of glycoside synthesis60, biocatalytic methods are lacking for C-nucleoside synthetic chemistry.To advance C-glycosidase for C-nucleoside synthesis. The enzyme’s natural role is the recycling of Ψ via degradation of its 5′-phosphate into uracil (Ura(12)) and d-ribose 5-phosphate (d-Rib5P(1)), as shown in Supplementary Fig. 62. Despite early evidence suggesting applicability65, the reverse C-glycosidase reaction . We show that YeiN catalyzes the selective 5-β-C-glycosylation of Ura (12) and derivatives thereof from several pentose 5-phosphate substrates, including the 5-phosphate of d-ribose, 2-deoxy-d-ribose, 2-deoxy-2-fluoro-d-ribose, d-arabinose or d-xylose. The ΨMP and its corresponding sugar or nucleobase variants are obtained in a single-step, highly efficient transformation. Remarkably, although formally a reversion of hydrolysis, the overall enzymatic conversion benefits from the favorable thermodynamics of its central catalytic step, a carbon-carbon bond-forming (Mannich-like) addition. It thus proceeds with excellent synthetic yields (≥90%), even in water. Finally, we show incorporation of the enzymatically synthesized C-nucleotide triphosphates into RNA. Collectively, therefore, our study implements biocatalytic methodology for C-nucleotide synthesis, which can facilitate XNA engineering for synthetic biology applications and establish new routes to natural product C-glycosides.Here, we identify pseudouridine monophosphate (ΨMP) cil (Ura1) and d-rion Fig.  is unexpd-Rib5P (1) and Ura (12), we tested a series of sugar phosphates  and nucleobase analogs  as alternative substrates , d-xylose 5-phosphate , d-arabinose 5-phosphate  and 2-deoxy-2-fluoro-d-ribose 5-phosphate  are active. l-Ribose 5-phosphate , d-glucose 6-phosphate , d-mannose 6-phosphate , d-fructose 6-phosphate , d-ribulose 5-phosphate  and d-erythrose 4-phosphate  are inactive. We also considered the 5-phosphates of 2-amino-2-deoxy- and 3-amino-3-deoxy-d-ribose, l-arabinose, d-lyxose, and l-xylose , 6-amino-Ura (14), 4-thio-Ura (15) and 2-thio-Ura (16) are active. Absence of activity with 1-methyl-Ura (17), 5-fluoro-Ura (18) and 6-trifluoro-methyl-Ura (19) can be mechanistically relevant, as discussed below. Additionally, thymine , 6-aza-Ura (21), 6-Cl-Ura (22), cytosine (23), 2,4-thio-Ura (24), 5-ethynyl-Ura (25), maleimide (26), adenine (27), guanine (28), 4-pyrimidone (29) and pyrimidine (30) were evaluated but found to be inactive. Combinatorial assessment of all sugar 5-phosphates active with Ura (12) against the full panel of Ura analogs used with d-Rib5P (1) confirms the substrate scope of YeiN  and Ura (12), catalytic efficiencies (kcat/KM) for alternative substrates are decreased between ~10 to ~103-fold, reflecting substantial (≥5-fold) changes in both kcat (decrease) and KM (increase).We obtained kinetic parameters for the substrates Fig. . Compared-Rib5P (1) results in a potentially catalytic complex, with the ε-amino group of Lys166 positioned (3.3 Å) for nucleophilic attack at the C1 carbonyl carbon. Thr139 (2.6 Å) and Glu31 (3.2 Å) are placed suitably to stabilize negative charge developing on the C1 oxygen atom during carbinol intermediate formation , the docking poses for d-Ery4P (11), d-Rul5P (10), and d-Fru6P (9) have Thr139 and Glu31 not properly aligned to the carbonyl oxygen, geometrically  and d-Rul5P (10) suggest loss of the stabilizing interaction with Gly132. The d-Glc6P (7) pose  into the structure of the covalent enzyme-d-Rib5P adduct (iminium ion form) places the reactive sugar C1′ and nucleobase C5 at a distance (3.4 Å) and orientation   Figs.  and 3c.12) C5 as a nucleophile of the reaction. The crystal structure of a covalent intermediate between YeiN and ΨMP shows just a single hydrogen bond between the C2 oxygen atom of Ura and Asn289  suggests additional polar contacts . Further docking analysis summarized in Supplementary Fig. d-Rib5P (1) can reflect steric restraint on the productive binding of certain nucleobases, such as 6-trifluoromethyl-Ura (19), 6-Cl-Ura (22) and 1-methyl-Ura (17)  analogs, including 5-fluoro-Ura (18) as well as 5-methyl-Ura (20) and 6-aza-Ura (21) . Positioned properly for catalysis, these Ura (12) analogs seem to be rendered inactive intrinsically. The proposed path for the catalytic reaction of YeiN  is an unlikely nucleophile for reaction with the iminium ion of the covalent enzyme intermediate. In the subsequent catalytic step, as shown in Fig. 12) analogs bearing substituents at the C5 are chemically incompetent to release the lysine analogously.Except for positioning, there seems to be limited catalytic facilitation from the enzyme to the agency of the Ura  and β-glycosidic (84%) forms during C-ribosylation of the naphthoquinone prealnumycin from d-Rib5P (1), suggesting ribosyl ring closure without enzymatic assistance  and Ura (12). The time-resolved NMR spectra for the synthesis direction  show that only a single anomer (β-C-riboside) is formed. They also show that solvent deuterium is incorporated at the Ura (12) C5 at a rate (0.07 mM/min) about 3-fold lower than the rate of conversion of Ura (12) into ΨMP (0.22 mM/min). This suggests that the deuterium uptake does not happen immediately during ΨMP synthesis but occurs only later when ΨMP and Ura (12) equilibrate dynamically. Suitable controls show that deuterium uptake into Ura (12) necessitates ΨMP formation  do not promote deuterium uptake into the C5 of Ura (12). Analyzing the hydrolysis of ΨMP  released from ΨMP incorporates solvent deuterium at its C5. The hydrolysis reaction approaches equilibrium after about 100 min and an equilibrium constant of 8.9 × 10−5 M can be calculated from the data .In the last step of the synthetic reaction, cyclization to a β-ion Fig. . The cycycosidic 4% forms ΨMP Fig. , we confd-Rib5P (1) released from ΨMP involves α-furanose (33%) and β-furanose (67%) forms at anomeric equilibrium. Plausible interpretation is that the open-chain form of d-Rib5P is released into solution where the furanose structures are formed spontaneously. However, the mutarotation of d-Rib5P (1)73 occurs at the limits of temporal resolution of the experiment, leaving the conformation of the released d-Rib5P (1) undecided.The NMR analysis Fig.  additionC-nucleoside monophosphates can arise from the requirement for a pentose 5-phosphate as the substrate. The unphosphorylated pentose, however, is an attractive starting material for synthesis. We therefore designed an enzymatic cascade reaction  and demonstrate applicability of the biocatalytic system to the synthesis of all sugar and nucleobase variants of the C-glycoside that were shown in Fig. 75.We considered that limitation on the synthetic utility of the YeiN-catalyzed formation of ion Fig.  in whichand Ura 1 and demoC-nucleoside or the C-nucleoside triphosphate. We therefore show enzymatic dephosphorylation of ΨMP with non-specific phosphatase to obtain the C-nucleoside in excellent yield , we set up a cascade reaction in which the monophosphate is phosphorylated by nucleotide monophosphate kinase and pyruvate kinase in the presence of ATP and PEP  and 6-amino ΨTP in a one-pot reaction starting from unphosphorylated sugar and Ura (12)  Fig. . ProductUsing the YeiN-encoding gene as the template, we examined amplification of the corresponding RNA and DNA using ΨTP, 6-amino-ΨTP and dΨTP in place of, or together with, the native UTP and dTTP, respectively. Applying T7 RNA polymerase as shown in Fig.2+ used. 6-Amino-ΨTP and dΨTP were not accepted by the used RNA polymerase. Applying DreamTaq-polymerase that is known for its relaxed dNTP substrate specificity, we observed that dΨTP cannot substitute for dTTP  was similar when ΨTP was used to substitute UTP. Unexpectedly, the band intensity decreased to about half when ΨTP and UTP were added at the same time. Overloading of the RNA polymerase with NTP substrate may have caused inhibition, perhaps due to limitation in the concentration of MgTTP Fig. . HoweverC-glycosidase for efficient biocatalytic synthesis of C-nucleoside monophosphates. Besides the natural ΨMP, a panel of sugar and nucleobase analogs of the C-nucleoside monophosphate are synthesized with excellent regiocontrol and stereoselectivity. The substrate requirements of the enzymatic reaction are consistent with the mechanistic proposal  and different naphthoquinones , d-2-deoxy-ribose (d-dRib), d-xylose (d-Xyl), d-arabinose (d-Ara), d-mannose (d-Man), d-glucosamine (d-GlcN), N-acetyl-d-glucosamine (d-GlcNAc), d-galactose , d-galactosamine , d-lyxose (d-Lyx), l-arabinose (l-Ara), l-xylose (l-Xyl), d-ribose-5-phosphate (d-Rib5P(1)), d-fructose 6-phosphate d-Fru6P(9), d-mannose 6-phosphate (d-Man6P (8)), d-glucose 6-phosphate (d-Glc6-P (7)), d-erythrose 4-phosphate (d-Ery4P (11)), d-ribulose 5-phosphate (d-Rul5P (10)), uracil (Ura (12)), 6-amino-Ura (14), 4-thio-Ura (15), 2-thio-Ura (16), 6-trifluoromethyl-Ura (19), 1-methyl-uracil (17), pyrimidine (29), pyrimidone (30), maleimide (26), adenine (27), guanin (28), thymine (20), and cytosine (23) were from Sigma Aldrich . 3-Methyl-Ura (13), 6-aza-Ura (21), 6-Cl-Ura (22), and l-ribose 5-phosphate (6) were from Carbosynth . 2-Amino-2-deoxy-d-ribose (2A-d-Rib), 3-amino-3-deoxy-d-ribose (3A-d-Rib), and 2-deoxy-2-fluoro-d-ribose (2F-d-Rib)) were from Synthose . Expression vectors (pet15b) containing the genes for ribokinase (RbsK) or β-pseudouridine 5′-phosphate glycosidase (YeiN) were obtained from Genescript . Restriction enzymes, Phusion polymerase, T7 RNA Polymerase, Dream Taq DNA polymerase, and GenJET® PCR clean-up kit were from Thermo Fisher Scientific . DNA sequencing was done at LGC Genomics .ATP ( > 95% purity) was from Roth . Deuterium oxide (99.96%) was from Euriso-Top . Calf intestine phosphatase (CIP) was from New England Biolabs . Rabbit muscle pyruvate kinase (PK), adenosine triphosphate (ATP), N-terminally His6-tagged E. coli YeiN was used. The expression vector (pet15_yein) as obtained from Genescript was transformed into the E. coli BL21_DE3 (pLys) expression strain by electroporation and transformants were selected on 0.1 mg/mL ampicillin LB-agar plates.An 600 of 0.1. At an OD600 of 0.8, the temperature was decreased to 18 °C and gene expression was induced with 0.4 mM IPTG  for 20 h. Cells were harvested by centrifugation at 2000 × g at 4 °C for 30 min using a Sorvall RC-5B refrigerated superspeed centrifuge . The supernatant was discarded, and the pellet was suspended in 50 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 8.0). Cells were disrupted using sonication  on ice and the cell-free supernatant was recovered by centrifugation at 4 °C and 2000 × g for 30 min. Pre-treated cell lysate (20 mL) was loaded on 2 × 5 mL HisTrap FF column , equilibrated with buffer A  and mounted on an ÄKTA prime plus  system. Protein was eluted using an imidazole gradient from 0% to 80% buffer B . The temperature was 10 °C and a flow rate of 3 mL/min was used. Fractions containing the target protein were pooled, concentrated and buffer-exchanged with Amicon Ultra-15 Centrifugal Filter Units . The final protein concentration was 30 mg/mL in 50 mM HEPES buffer containing 2 mM MgCl2 (pH 7.0). Enzyme was stored at −20 °C until further use. Protein purification was monitored by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS PAGE) . About 1.5 µM YeiN was used in the reaction. The substrates used were d-Rib5P (1) (2 mM) and Ura (12) (1 mM). One unit of YeiN activity is the amount of enzyme producing 1 μmol of ΨMP/min under the conditions used. Enzyme preparations had typically a specific activity of ∼3 U/mg.The YeiN activity assay was performed at 37 °C in 50 mM HEPES buffer supplemented with 2 mM MnClE. coli BL21_DE3 (pLys) expression strain by electroporation and transformants were selected on 0.1 mg/mL ampicillin LB-agar plates. RbsK was expressed and purified as described for YeiN, except the protein was stored in 50 mM HEPES supplemented with 2 mM MgCl2 (pH 7.0).The expression vector (pet15_RbsK) as obtained from Genescript was transformed into the N-terminally His6-tagged E. coli RbsK was used. The RbsK activity assay was performed at 37 °C in 50 mM HEPES buffer  containing 2 mM MgCl2 and 3 µM RbsK. The substrates used were ATP (10 mM) and d-Rib (10 mM). One unit of RbsK activity is the amount of enzyme producing 1 μmol of d-Rib5P (1)/min under the conditions used. The enzyme preparation had a specific activity of ∼50 U/mg.An Bacteroides thetaiotaomicron VPI-5482 was used as an N-terminal fusion with the positively charged binding module Zbasic288. Expression, harvest and cell disruption was performed as described for YeiN except 50 µg/mL kanamycin was used instead of ampicillin.The sugar phosphate phosphatase BT4131 from 2 (pH 7.0). The enzyme was stored at 4 °C until further use. Protein purification was monitored using SDS PAGE.Cell-free extract (20 mL) was loaded on 2 × 5 mL HiTrap SP FF column , equilibrated with buffer A  and mounted on an ÄKTA prime plus  system. Protein was eluted using an NaCl gradient from 0% to 100% buffer B  at 10 °C using a flow rate of 3 mL/min. Fractions containing the target protein were pooled, concentrated and buffer-exchanged with Amicon Ultra-15 Centrifugal Filter Units . The final protein concentration was 10 mg/mL in 50 mM HEPES containing 2 mM MgCl2 (pH 7.0) at 37 °C using 20 mM d-Glc6P as the substrate. The reaction was started with 1.5 µM BT4131 and samples taken were quenched by heat treatment. Inorganic phosphate was measured at 850 nm88. One unit of BT4131 activity is the enzyme amount releasing 1 μmol of phosphate from d-Glc6P/min under the conditions applied. The specific activity of the phosphatase preparation was typically ∼16 U/mg.The specific activity was determined in 50 mM HEPES buffer supplemented with 5 mM MgCl(umpk) and cytidine monophosphate kinase (cmpk) genes were amplified from genomic E. coli BL21_DE3 (pLys) DNA by PCR using Phusion polymerase and the following set of primers (restriction sites underlined): UMPK_NdeI_fw (CATATGGCTACCAATGCAAAACCCGTC), UMPK_XhoI_rv (CTCGAGTTATTCCGTGATTAAAGTCCCTTCTTTTTC); CMPK_NdeI_fw (CATATGACGGCAATTGCCCCGGTTATTACC), CMPK_XhoI_rv (CTCGAGTTATGCGAGAGCCAATTTCTGGCGC). Doubly digested (NdeI and XhoI) PCR products were purified from agarose gels treated with shrimp alkaline phosphatase  and ligated (T4 ligase) into the linearized (NdeI and XhoI) pET-STREP3 expression vector. The constructed vector was transformed into electro-competent E. coli BL21_DE3 (pLys) and transformants were selected on LB-agar plates (0.05 mg/mL kanamycin). Isolated vectors were sequenced (LGC Genomics). NPK expression, cell harvesting and cell disruption was performed as described for YeiN, except that 0.05 mg/mL kanamycin instead of ampicillin was used and cells were dissolved in wash buffer . Cell-free extract (20 mL) was loaded (2 mL/min) on 1 × 5 mL StrepTrap HP column , equilibrated with wash buffer and mounted on an ÄKTA prime plus  system. Unspecifically bound protein was washed off with 10 column volumes of wash buffer at a flow of 3 mL/min. Protein was eluted using elution buffer . Fractions containing the target protein were pooled, concentrated and buffer-exchanged with Amicon Ultra-15 Centrifugal Filter Units (Millipore). The final protein concentration was 15 mg/mL in 50 mM HEPES containing 2 mM MgCl2 (pH 7.0). Enzyme was stored at −20 °C until further use. Protein purification was monitored using SDS PAGE  containing 1 mM MgCl2 and 5 µM CMPK. The substrates used were 3 mM UMP and 3 mM ATP. One unit of NPK activity is the amount of enzyme producing 1 μmol of UDP/min under the conditions used. CMPK preparation typically exhibited a specific activity of 20 U/mg.An N-terminally Strep-tagged variant was used. The ndk gene was amplified from genomic E. coli BL21_DE3 (pLys) DNA by PCR using Phusion polymerase and the following set of primers (overlapping 5'-overhangs in lower case letters): NDK_fwd (ATGGCTATTGAACGTACTTTTTCCATC), NDK_rv (TTAACGGGTGCGCGGGC). The PCR product was cloned into the pET-STREP3 expression vector by circular polymerase extension cloning. Briefly, using the primer pairs CEPEC_NDK_fwd (GTTCGAGAAAGGCTTAATTAACCATATGgctattgaacgtactttttccatc), CPEC-NDK_rv (GTGGTGGTGGTGGTGCTCGAGttaacgggtgcgcgggc) and CPEC-NDK-Strep_fwd (ggaaaaagtacgttcaatagcCATATGGTTAATTAAGCCTTTCTCGAAC) CPEC-NDK-Strep_rv (gcccgcgcacccgttaaCTCGAGCACCACCACCACCAC) in two independent PCR reactions, complementary overlaps are introduced into the ndk gene and into the target vector.An E. coli XL1 Blue. Transformants were selected on LB-agar plates containing 0.1 mg/mL of ampicillin, and the isolated plasmid was sequenced. The sequenced vector was transformed into the expression strain E. coli BL21_DE3 (pLys) and transformants were selected on LB-agar plates containing 0.1 mg/mL of ampicillin. NDK expression and purification were as described for NDKs  and 3 mM ATP. One unit of NDK activity is the amount of enzyme producing 1 μmol of UTP/min under the conditions used. The enzyme preparation had a specific activity of 21 U/mg.The NDK activity assay was performed at 37 °C in 50 mM HEPES buffer (pH 7.0) containing 1 mM MgClhttps://web.expasy.org/protparam/). RbsK , YeiN , CMPK , NDK , BT4131 .Protein concentrations were measured using DS-11 Spectrophotometer  at 280 nm. Protein concentration was calculated using the corresponding molar extinction coefficient and the molecular weight computed by protparam (12)  mM−1 cm−1), 4-thio-Ura (15) , 2-thio-Ura (16) , thymidine (20) , guanine (28) , cytosine (23) , adenine (27) .Uracil content was measured photometrically using a DS-11 Spectrophotometer (DeNovix). Ura (12) (0.3 min), ΨMP (0.4 min), ΨDP (0.9 min), ADP (1.1 min), ΨTP (2.7 min), and ATP (4 min).Nucleosides were quantified by reversed-phase ion-pairing HPLC. Typically, 5 µL of sample containing an overall nucleoside concentration of about 1 mM were loaded on a Kinetex C18 column . Analytes were separated in 5-min long isocratic runs using 20 mM phosphate buffer, pH 5.9, containing 40 mM tetra-n-butylammonium bromide (TBAB) and 12.5% acetonitrile. The flow rate was 2 mL/min and the temperature set to 35 °C. Eluents were detected at 260 nm. A 5-min HPLC trace is shown in Supplementary Fig. 2O (2:1:1). The development solution consisted of 0.5 g thymol, 95 mL EtOH and 5 mL H2SO4. Reducing sugar spots were developed by heating the plate to approximately 70 °C for 2 min using a heat-gun . The mobile phase was composed of 2-BuOH:AcOH:Hd-Rib, d-dRib, d-Xyl, d-Ara, 2F-d-Rib, d-Glc, 2F-d-Rib, 3A-d-Rib, l-Xyl, l-Ara, d-Lyx, d-Man, d-GlcN, d-GlcNAc, d-GlcA, d-Gal, d-GalN), 100 mM PEP, 2 mM ATP and 10 mM HEPES buffer (pH 7.0) containing 1 mM MgCl2 and enzymes  in a total volume of 100 µL. The reactions were incubated at 37 °C without agitation for 20 h and 5 µL sample were analyzed by TLC. Of the tested sugars, d-Rib, d-dRib, d-Xyl, d-Ara,2F-d-Rib, d-Glc were accepted.Prior to synthesis, the substrate scope of RibK was evaluated using mixture of 100 mM sugar (d-Rib 5-phosphate (2F-D-Rib5P (5) that was prepared in a 1 mL reaction. Reaction mixtures contained 100 mM pentose , 100 mM PEP, 2 mM ATP in 10 mM HEPES buffer (pH 7.0) with 1 mM MgCl2 and 0.4 µM PK. For the phosphorylation of d-Rib, 15 µM RbsK, for d-dRib 30 µM RbsK, and for other pentoses 60 µM RbsK were used. The reactions were incubated at 37 °C without agitation for 20 h and 5 µL sample were analyzed by TLC. The reaction was stopped by heat treatment at 99 °C for 10 min and precipitated protein was removed by centrifugation for 10 min at 4 °C and 16,000 × g. The reactions were diluted with twice volume of water and applied  to a FliQ FPLC column  packed with SuperQ-650M  anion exchange resin mounted onto an ÄKTA prime plus FPLC system . The column was equilibrated in 10 mM sodium acetate (NaAc), pH 4.5. Products were eluted using a linear gradient of 1 M NaAc (pH 4.5), from 0% to 50% in 120 mL with a flow of 4 mL/min. Eluting fractions were evaluated for the presence of sugar phosphates using TLC. Following the product elution, the column was regenerated with 100% of 1 M NaAc buffer for 10 min. The fractions containing pentose 5-phosphates were pooled and concentrated under reduced pressure  to a total volume of 1 mL using a rotary evaporator . The concentration of pentose 5-phosphates were evaluated as follows: sugar phosphates were completely hydrolyzed by applying 10 µM of sugar phosphate phosphatase (AGP)88 for 20 h. The released inorganic phosphate was determined in a colorimetric assay based on the formation of a blue phospho-molybdate complex at 850 nm89.Synthesis was performed at 5 mL scale, except for 2-deoxy-2-fluoro-2 in a total volume of 100 μL. Nucleobase screening was performed by incubating 5 mM d-Rib5P (1) and 1 mM of nucleobase (Ura (12), 3-methyl-Ura (13), 6-amino-Ura (14), 2-thio-Ura (15), 4-thio-Ura (16), 1-methyl-Ura (17), 5-fluoro-Ura (18), 6-trifluoro-methyl-Ura (19), 6-aza-Ura (21), 6-Cl-Ura (22), 2,4-dithio-Ura (24), 5-ethynyl-Ura (25), maleimide (26), cytosine(23), thymine (20), adenine (27), guanine (28), 4-pyrimidone (29) and pyrimidine (30)) with 30 µM YeiN. Sugar phosphates were screened by incubating 1 mM Ura and 5 mM of sugar phosphate (d-Rib5P (1), d-dRib5P (2), d-Xyl5P (3), d-Ara5P (4), d-Fru6P (9), d-Man6P (8), d-Glc6P (7), d-Rul (10), d-Ery4P (11), 2F-d-Rib5P (5), l-Rib5P (6) with 30 µM YeiN. Samples (20 µL) were withdrawn after 0, 3, 24 h and quenched by methanol addition (100 µL). Precipitated protein was removed by centrifugation at 20,000 × g for 5 min and supernatant was analyzed by HPLC.All reactions were performed at 37 °C in 50 mM HEPES buffer (pH 7.0) containing 2 mM MnCld-Rib5P (1), d-dRib5P (2), d-Xyl5P (3), d-Ara5P (4), d-2F-Rib5P(5)) were incubated with 2 mM of each nucleobase (Ura (12), 3-methyl-Ura (13), 6-amino-Ura (14), 2-thio-Ura (15), 4-thio-Ura (16), 1-methyl-Ura (17), 5-fluoro-Ura (18), 6-trifluoro-methyl-Ura (19), 6-aza-Ura (21), 6-Cl-Ura (22), 2,4-dithio-Ura (24), 5-ethynyl-Ura (25), maleimide (26), cytosine(23), thymine (20), adenine (27), guanin (28), 4-pyrimidone (29) and pyrimidine (30)) and 30 µM of YeiN for 3 h. The reactions were quenched by methanol addition (500 µL) and precipitated protein was removed by centrifugation at 20,000 × g for 5 min prior to HPLC analysis.Five millimolar of pentose phosphates (previously isolated as described above) accepted by YeiN in the initial screen  in 50 mM HEPES (pH 7.0) supplemented with 2 mM MnCl2. Reactions were incubated at 37 °C on a Thermomixer comfort  with agitation of 300 rpm. Initial rates for ΨMP synthesis with nucleobases (Ura (12), 6-amino-Ura (13), 4-thio-Ura (14), 2-thio-Ura (15), 3-methy-Ura(16)) were determined at a constant d-Rib5P (1) concentration of 5 mM and at nucleobase concentrations between 0.05 mM and 1.00 mM. Initial rates for ΨMP synthesis with pentose 5-phosphates (d-Rib5P (1), d-dRib5P (2), d-Ara5P (4), d-Xyl5P(3) were determined at a constant Ura (10) concentration of 5 mM and 1.00−0.05 mM of the corresponding pentose 5-phosphate. Reactions were started by YeiN (1.00 µM−100 µM) added in a concentration adjusted to the enzyme’s specific activity with the substrate used. The reaction mixture was incubated for 20 min and quenched by methanol addition . Precipitated protein was removed by centrifugation at 20,000 × g for 5 min and samples were analyzed by HPLC. It was ensured that conversion was below 20%. Kinetic parameters were obtained from non-linear least squares fits  of initial ΨMP-synthesis rates to Michaelis–Menten Eq. , 100 mM PEP, 2 mM ATP and 10 mM HEPES buffer (pH 7.0), 1 mM MgCl2 and 0.4 µM pyruvate kinase. For the phosphorylation of d-Rib 15 µM RbsK, for d-dRib 30 µM RbsK and for d-Ara and d-Xyl 60 µM RbsK was used. Reactions were performed in a total volume of 5 mL. Samples (5 µL) were taken at reaction start and then after 2 h and 20 h. They were analyzed by TLC.Step one: pentose phosphorylation. The reaction mixture contained 100 mM pentose , d-dRib5P (2), d-Xyl5P (3), d-Ara5P(4)) using 10 mM HEPES (pH 7.0). Ten mM of nucleobase (Ura (12), 6-amino-Ura (13), 2-thio-Ura (14), 4-thio-Ura (15), 3-methyl-Ura (16)) were added to the phosphorylation reaction. ΨMP-synthesis was started with 15 µM YeiN. To compensate for the lower activity of YeiN towards different pentose 5-phosphates and nucleobases, the YeiN concentration was adjusted as follows: for the synthesis of dΨMP 30 µM YeiN was used; for synthesis of Ara-ΨMP, Xyl-ΨMP, 6-amino-ΨMP, 2-thio-ΨMP and 4-thio-ΨMP, 90 µM YeiN was used. Samples (5 µL) were taken at start and after 5, 15, 30, 60, 120, 180, 240, 300, 360 min, and 10 h for 2-thio-ΨMP, Ara-ΨMP, Xyl-ΨMP. Each sample was quenched by 100 µL methanol. Precipitated protein was removed by centrifugation for 20,000 × g for 5 min and the sample was analyzed by HPLC. After completion of the reaction or latest after 10 h of incubation, the reactions were stopped by heat inactivation for 5 min at 99 °C. Precipitated protein was removed by centrifugation for 10 min at 4 °C and 16,000 ×  g.Step two: 2 and incubated with 2 U/mL of CIP at 37 °C for 600 min. Ten microliters samples were withdrawn at start, and after 30, 90, 600 min, and quenched in 100 µL methanol. Precipitated enzyme was removed by centrifugation  and supernatant was analyzed by HPLC.ΨMP (5 mM) was dissolved in 15 mL 10 mM HEPES buffer, pH 8.0, supplemented with 1 mM MgCl2, 30 µM PK, 12 mM PEP, 50 mM HEPES, pH 7.0, in a total volume of 250 µL. Ten µL samples taken at certain times were quenched in 100 µL methanol. The precipitated enzyme was removed by centrifugation  and supernatant was analyzed by HPLC. To test the effect of NDK removal for ΨTP-synthesis the same reaction set up without addition of 40 µM NDK was used , 6-amino-Ura (13), 2-thio-Ura (14), 4-thio-Ura (15), 3-methyl-Ura (16)), 1 mM MnCl2, and 10 mM HEPES buffer were added to the reaction mixture to a final concentration of 25 mM pentose 5-phosphate. The reaction was started with YeiN . ΨMP phosphorylation was started with 15 mM PEP, 15 µM CMPK, and 1 mM ATP, resulting in a volume increase of 33%. Ten microliters of samples were quenched by addition of 100 μL methanol and precipitated protein was removed by centrifugation for 20,000 × g for 5 min prior to HPLC analysis. Enzymes were heat inactivated  after 21 h reaction and precipitated protein was removed by centrifugation for 10 min at 4 °C and 16,000 × g.Pentose phosphorylation was performed as described above under “Step one: pentose phosphorylation”. For ΨMP synthesis, 15 mM nucleobase  and precipitated protein was removed by centrifugation for 10 min at 4 °C and 16,000 ×  g.BT4131 (10 µM) and 1 mM MgClC-nucleoside were pooled and concentrated under reduced pressure  to a total volume of 10 mL using a rotary evaporator .Enzyme-free reaction mixture was applied  to a FliQ FPLC column  packed with SuperQ-650M  anion exchange resin mounted onto an ÄKTA prime plus FPLC system . The column was equilibrated in 10 mM NaAc, pH 4.5. Products were eluted using a linear gradient of 1 M NaAc, pH 4.5, from 0% to 50% in 120 mL with a flow of 4 mL/min and monitored at 260 nm. Following product elution, buffer B was set to 100% for 10 min to elute unspecifically bound substances. The fractions containing the desired 2O and mounted onto an ÄKTA prime plus FPLC system . Compound elusion was performed using doubly distilled water and monitored at 260 nm. Fractions containing salt-free product were collected, pooled and concentrated under reduced pressure  to a total volume of 10 mL using a rotary evaporator  and lyophilized.To remove excess NaAc, concentrated products were split in 5 × 2 mL portions and loaded onto a size exclusion column  packed with Sephadex G10 (exclusion limit <700 Da) equilibrated in double distilled H90 as implemented in Yasara v. 18.2.7 was used for enzyme-ligand docking. The AmberFB-1591 force field and the default parameters provided by the standard docking macro were used. The structure of YeiN (pdb code: 4GIL)62 was used as a receptor in molecular-docking experiments. For dockings of sugar phosphates (d-Rib5P (1), d-Rul5P (10), d-Fru6P (9), d-Glu6P (7), d-Ery4P(11)), the ring-opened intermediate was deleted and a simulation cell of 15 Å × 10 Å × 10 Å was placed at the former location of d-Rib5P (1). Sugar phosphates were docked in the ring-open conformation. The protonation state of key catalytic residues  was set prior to the docking experiment. The protonation states of all other protein residues were set automatically for a pH of 7.00. For dockings of nucleobases (Ura (12), 6-trifluoro-methyl-Ura (19), 6-Cl-Ura (22), 6-aza-Ura (21), 1-methyl-Ura (17), 5-fluoro-Ura (18), 5-methyl-Ura(20)), the uracil moiety was deleted and the charge of lysine was set to fit the covalent iminium intermediate. The protonation states of all other protein residues were set automatically for a pH of 7.00. A simulation cell of 15 Å × 10 Å × 10 Å was placed at the former location of Ura (11). Following the docking runs, the obtained poses were optimized by energy using the standard macro included in Yasara. All ligands were generated using the Grade Web Server 92. Docking poses were evaluated by their associated free energy and mechanistic plausibility.Molecular docking was used to structurally evaluate the substrate scope of YeinN. AutoDock13yein gene was amplified from the YeiN expression vector (peT15_yein) by PCR using Phusion polymerase and a set of primers complementary to the T7 promoter (TAATACGACTCACTATAGGG) or terminator (GCTAGTTATTGCTCAGCGG). The PCR product was purified via column purification  and eluted in DEPC-water. IVT was performed using 30 U of T7 RNA polymerase, 600 ng template DNA, Transcription Buffer , 6 mM MgCl2, 10 mM DTT, 10 mM NaCl and 2 mM spermidine) and 2 mM of each NTP  in a total volume of 50 µL. Synthesis of modified RNA was performed in two ways; either 2 mM of ΨTP  was additionally added to the conventional NTPs or, UTP was fully replaced by 2 mM of either ΨTP, dΨTP or 6-amino-ΨTP. The reaction was incubated at 37 °C in the thermocycler  and the resulting PCR products were evaluated by agarose gel electrophoresis.In vitro translation (IVT) using T7 RNA polymerase (Thermo Fisher Scientific) was performed following the manufacturer’s protocol. Shortly, linear DNA template containing the T7 promoter and the yein gene was amplified from 50 ng expression vector (pet15_yein) using 1.25 U of Dream-Taq DNA polymerase, 0.2 mM of dNTPs , 0.5 µM T7_fwd, 0.5 µM T7_bwd primers and DreamTaq Buffer as recommended by the supplier (Thermo Fisher Scientific). To incorporate Ψ into the DNA either 0.2 mM of ΨTP were additionally added to dNTP mix or fully replaced dTTP by dΨTP. The temperature profile consisted of a preheating step at 95 °C for 1 min, followed by 30 reaction cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 2 min. The final extension step was carried out at 72 °C for 5 min. PCRs were performed using the Doppio-Dual thermocycler (VWR). The resulting PCR products were evaluated by agarose gel electrophoresis.To evaluate the potential of PCR catalyzed generation of pseudouridinated DNA, the d-Rib5P (1) and Ura (12), as well as hydrolysis of ΨMP, were analyzed by 1H-NMR. All reagents including the enzyme were dissolved in D2O and the reaction was set up in a 5 mm high-precision NMR sample tube . YeiN was buffered by washing the enzyme with 10-fold excess of 10 mM potassium phosphate buffer (pD 7.5) supplemented with 0.1 mM MnCl2, using centrifugation  with Vivaspin 500 centrifugal concentrators (10 kDa cut-off). The synthesis reaction contained 14 mM Ura (12) and 8 mM d-Rib5P (1), while 10 mM ΨMP were used for the hydrolysis reaction. Each reaction was initiated by adding 3 µM enzyme. The reactions were incubated at 30 °C without agitation in the electromagnetic field.YeiN-catalyzed synthesis of ΨMP from 1H-NMR spectra (499.98 MHz) were recorded on a 5 mm indirect detection PFG-probe with pre-saturation of the water signal by a shaped pulse. The following standard pre-saturation sequence was used: 2 s relaxation delay; 90° proton pulse; 2.048 s acquisition time; 8 kHz spectral width; number of points 32 k.Measurements were performed with a Varian INOVA 500-MHz NMR spectrometer . The VNMRJ 2.2D software was used for all measurements. 12) without transient formation of ΨMP was evaluated by 1H-NMR. All reagents including the enzyme were dissolved in D2O and prepared as described above. The 600 µL reactions contained 5 mM Ura (12) and 5 mM of sugar phosphate (d-Rib5P (1), d-Ery4P (11), d-Rul5P (10), d-Glc6P (7), d-Fru6P(9)) in 10 mM potassium phosphate buffer (pD 7.5) supplemented with 0.1 mM MnCl2. Conversions were started by addition of 3 µM enzyme and incubated at 30 °C in a Thermomixer comfort  with agitation of 300 rpm for 3 h. The reactions were stopped by heat treatment  and precipitated protein was removed by centrifugation for 10 min at 4 °C and 16,000 × g. The supernatant was transferred into a 5 mm NMR sample tube and analyzed by 1H-NMR.The potential of YeiN-catalyzed deuterium wash-in into Ura (1H-NMR spectra (499.98 MHz) were measured on a 5 mm indirect detection PFG probe. Standard pre-saturation sequence was used: relaxation delay 2 s; 908 proton pulse; 2.048 s acquisition time; spectral width 8 kHz; number of points 32,000 13C NMR spectra (125.71 MHz). Standard pre-saturation sequence was used: relaxation delay 2 s; 908 proton pulse; 2.048 s acquisition time; spectral width 8 kHz; number of points 32 k. MNova 12.0.3  was used for data processing and analysis  in a total volume of 600 μL and transferred to a 5 mm high-precision NMR sample tube. They were analyzed using a Varian INOVA 500-MHz NMR spectrometer (Agilent Technologies). The VNMRJ 2.2D software was used for measurements. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


N-{methyl}benzamides, which crystallized as ethanol monosolvates, the independent components are linked by hydrogen bonds to form centrosymmetric four-molecule aggregates.In each of the title newly synthesized and closely related  N-{methyl}benzamides have been prepared by acid-promoted condensation reactions between 3-aryl-4-formylsydnones and N-[methyl]benzamide, and both have been crystallized as ethanol monosolvates. N-{methyl}benzamide ethanol monosolvate, C19H15N7O3S·C2H6O (I), and N-({4-[3-(4-methylphenyl)-4-sydnonylideneamino]-5-sulfanylidene-1H-1,2,4-triazol-3-yl}methyl)benzamide ethanol monosolvate, C20H17N7O3S·C2H6O (II), differ only in the presence of a methyl group for (II) instead of a hydrogen atom for (I), and in both of them the ethanol component is disordered over two sets of atomic sites having occupancies of 0.836 (6) and 0.164 (6) in (I), and 0.906 (6) and 0.094 (6) in (II). Combinations of O—H⋯O and N—H⋯O hydrogen bonds link the molecules into cyclic, centrosymmetric four-molecule aggregates. Comparisons are made with the structures of some related compounds.Two new  H)-one] system have been shown to exhibit a wide range of biological activities, including analgesic -5-sulfanylidene-1H-1,2,4-triazol-3-yl]methyl}benzamide (I) and N-({4-[3-(4-methylphenyl)-4-sydnonylideneamino]-5-sulfanylidene-1H-1,2,4-triazol-3-yl}methyl)benzamide (II). Compounds (I) and (II) were prepared using an acid-mediated condensation between the 3-aryl-4-formylsydnones (A) 2  Fig. 1 and the I) and (II) are closely related and differ only in the methyl group that is attached to C44 for (II) instead of a hydrogen atom for (I): each structure can readily be refined starting from the atomic coordinates of the other, provided that the necessary adjustment is made to the substituent at atom C44 , and 0.906 (6) and 0.094 (6) in (II).The molecular and crystal structures of compounds -thione form, as shown by the localization of the H atom on N21 in a difference-Fourier map and the subsequent refinement of its atomic coordinates, by the intermolecular hydrogen bonds (Tables 1I) and 1.661 (3) Å in (II). These values are typical for those found in thiones -1,3,4-oxadiazole-2-thione (IV)   chains, running parallel to [001] and [010], respectively, in the triclinic unit cells. Although no crystal structure has yet been reported for the intermediate (B)  and (II), the fact that all of compounds (I)–(V) crystallize in the thione form makes it seem likely that the intermediate also exists in this tautomeric form in the solid state, although it may well exist as an equilibrium mixture of thione and thiol (mercapto) forms in solution, with the position of equilibrium possibly differing from one solvent to another. However, it must be emphasized that, to date, no studies have been made of the constitution of this intermediate in solution. On the other hand, a masked form of the thiol tautomer is present in 2-{5-[methyl]-1,3,4-oxadiazol-2-ylthio}-1-ethanone (VI)  chains.It is of interest briefly to compare the structures of compounds (B) Fig. 1 used in et al., 2010N-[methyl]benzamide (B)  and (II), the appropriate intermediate (A)  was added to a solution of (B)  in ethanol (15 ml). Concentrated sulfuric acid (0.5 ml) was then added to each of these mixtures, under vigorous stirring, and stirring was then continued for 4 h. The resulting solid products were collected by filtration and then washed, first with ethanol and then with water, before being dried in air. Compound (I), yield 72%, m. p. 435 K, IR (cm−1) 3170 (NH), 1740 (C=O), 1660 (C=O), 1590 (C=N). Compound (II), yield 76%, m. p. 505 K, IR (cm−1) 3149 (NH), 1769 (C=O), 1665 (C=O), 1595 (C=N). Crystals of (I) and (II) suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature and in the presence of air, of solutions in ethanol/N,N-dimethylformamide mixtures .Previously published methods were used for the preparation of the 3-aryl-4-formylsydnones (A) Fig. 1 (Goh et 3) or 0.97 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were allowed to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms: the H atoms bonded to C atoms in the minor disorder components were included on the same basis. For the H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), giving the N—H distances shown in Tables 1Ueq(O). However, using the normal riding models for hydroxyl H atoms, it was not possible to establish satisfactory positions for these H atoms in the minor disorder components, and accordingly they were included in calculated positions, riding at 0.82 Å from the atoms O61, at positions calculated by interpolation along the O61⋯O1 vectors, again with Uiso(H) = 1.5Ueq(O). The refined occupancies for the disorder components were 0.836 (6) and 0.164 (6) in (I), and 0.906 (6) and 0.094 (6) in (II).Crystal data, data collection and refinement details are summarized in Table 310.1107/S2056989020007483/wm5563sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989020007483/wm5563Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020007483/wm5563IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2007902, 2007901CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In complexes 1–3, the corresponding nitrogen-containing heterocycle acts as a bridging ligand between two Ag(I) ions. In vitro antimicrobial activity of these complexes, along with the ligands used for their synthesis, was evaluated against the broad panel of Gram-positive and Gram-negative bacteria and fungi. The silver(I) complexes 1–3 showed selectivity towards Candida spp. and Gram-negative Escherichia coli in comparison to the other investigated bacterial strains, effectively inhibiting the growth of four different Candida species with minimal inhibitory concentrations (MICs) between 2.5 and 25 μg/mL and the growth of E. coli, with MIC value being 12.5 μg/mL. Importantly, complex 2 significantly reduced C. albicans filamentation, an essential process for its pathogenesis. Antiproliferative effect on the normal human lung fibroblast cell line MRC-5 was also evaluated with the aim of determining the therapeutic potential of the complexes 1–3. The interactions of these complexes with calf thymus DNA (ctDNA) and bovine serum albumin (BSA) were studied to evaluate their binding activities towards these biomolecules for possible insights on their mode of action.1,2-Bis(4-pyridyl)ethane (bpa) and 1,2-bis(4-pyridyl)ethene (bpe) were used for the synthesis of polynuclear silver(I) complexes, {[Ag(bpa)]NO The invasive microbial infections are seen as a rapidly increasing global threat to human health, in particular in immunocompromised patients, having an unacceptably high mortality rate despite the availability of antimicrobial drugs , 2. ThisN-heterocycles) in which two pyridine rings are linked by–CH2–CH2– and–CH═CH–groups, 1,2-bis(4-pyridyl)ethane (bpa) and 1,2-bis(4-pyridyl)ethene (bpe), respectively. These ligands have previously been used as linkers in the synthesis of metal-organic frameworks NO3}n (1), {[Ag(bpa)2]CF3SO3.H2O}n (2), and {[Ag(bpe)]CF3SO3}n (3), were synthesized using slightly different approach. The complexes were evaluated for their in vitro antimicrobial activity and the effect on the viability of a human normal fibroblast cell line (MRC-5). In order to gain an insight into the reactivity of complexes 1–3 with potential biological targets, their interactions with the calf thymus DNA (ctDNA) and bovine serum albumin (BSA) were examined.The crucial factors in determining the antimicrobial effectiveness of silver(I) complexes are the type of the ligand donor atom bound to the Ag(I) ion and the strength of the resulting Ag–donor bonds . Thus, sameworks , 23. Con3 and AgCF3SO3), 1,2-bis(4-pyridyl)ethane (bpa), 1,2-bis(4-pyridyl)ethene (bpe), ethanol, acetonitrile, dimethyl sulfoxide (DMSO), deuterated dimethyl sulfoxide (DMSO-d6), calf thymus DNA (ctDNA), Tris buffer (tris(hydroxymethyl)aminomethane), ethidium bromide (EthBr), and bovine serum albumin (BSA) were obtained from the Sigma-Aldrich. All chemicals were of reagent-grade quality or higher and used without further purification.The silver(I) salts (AgNO1H and 13C NMR spectra of the N-heterocyclic ligands and the silver(I) complexes were recorded at room temperature on a Varian Gemini 2000 spectrometer . 5.0 mg of each compound was dissolved in 0.6 mL of DMSO-d6 and transferred into a 5 mm NMR tube. Chemical shifts, δ, are reported in parts per million (ppm) and scalar couplings (J) are reported in Hertz. Chemical shifts were calibrated relative to those of the solvent. The abbreviations for the peak multiplicities are as follows: s (singlet), d (doublet), dd (doublet of doublets), and m (multiplet). In order to investigate the solution behavior of silver(I) complexes, the 1H NMR spectra were recorded immediately after their dissolution and after 48 h standing in the dark at room temperature. The UV-Vis spectra were recorded on a Shimadzu double-beam spectrophotometer after dissolving the corresponding silver(I) complex in DMSO over the wavelength range of 200–900 nm. The concentration of the silver(I) complexes was 2.36·10−4 (1), 4.31.10−5 (2), and 1.50.10−5 M (3). The IR spectra were recorded as KBr pellets on a PerkinElmer Spectrum 100 spectrometer over the wavenumber range of 450–4000 cm−1.The elemental analyses of the synthesized silver(I) complexes for carbon, hydrogen, and nitrogen were performed by the Microanalytical Laboratory, Faculty of Chemistry, University of Belgrade. The The method applied for the preparation of these complexes has been optimized in comparison to the previous reports , 25 and 3 for 1 and 256.9 mg of AgCF3SO3 for 2 and 3) in 5.0 mL of ethanol was added slowly under stirring to the solution containing 0.5 mmol of 1,2-bis(4-pyridyl)ethane  and 1,2-bis(4-pyridyl)ethene  in 5.0 mL of ethanol. A white precipitate was formed immediately after addition of the silver(I) salt. The reaction mixture was stirred in the dark at ambient temperature for 4 h, and then, the precipitate was filtered off and recrystallized in acetonitrile (1) or acetonitrile/water . The obtained solutions were left at room temperature, and after 4–6 days, colorless crystals of complexes 1–3 were formed. These crystals were filtered off and dried in the dark at ambient temperature. Yield  was 132.8 mg (75%) for 1, 114.2 mg (71%) for 2, and 184.4 mg (84%) for 3.The solution of 1.0 mmol of the corresponding silver(I) salt (169.9 mg of AgNO1 = C12H12AgN3O3 (MW = 354.12): found: C, 40.54; H, 3.51; and N, 11.69%; calc.: C 40.70; H, 3.42; and N, 11.87%. 1H NMR : δ = 2.98 , 7.32 , and 8.46  ppm. 13C NMR : δ = 34.57 (CH2), 124.31 (C3 and C5), 149.36 (C2 and C6), and 150.59 (C4) ppm. IR : 3030 w, 2925 w (ν(Car–H)), 2858w (ν(C–H)), 1606 s, 1559 w, 1499 w (ν(Car = Car) and ν(Car = N)), 1384 (νas(NO3)), 1221 m (ν(Car–N)), 826 s, 546 m (γ(Car–H)). UV-Vis : 257 (ε = 3.3·103 M−1·cm−1).Elemental analysis for 2 = C25H26AgF3N4O4S (MW = 643.43): found: C, 46.44; H, 4.01; and N, 8.59%; calc.: C 46.67; H, 4.07; and N, 8.71%. 1H NMR : δ = 2.96 , 7.29 , 8.45  ppm. 13C NMR : δ = 34.52 (CH2), 124.05 (C3 and C5), 149.57 (C2 and C6), 150.02 (C4) ppm. IR : 3429br (ν(O–H)), ∼3000 w (ν(Car–H)) and (ν(C–H)), 1615 s, 1560 m, 1507 w, 1434 m (ν(Car = Car) and ν(Car = N)), 1279vs, 1251vs (νas(SO3)), 1223 s (νs(CF3)), 1160 s (νas(CF3)), 1024vs (νs(SO3)), 831 m, 597 w (γ(Car–H)). UV-Vis : 257 (ε = 1.8·104 M−1·cm−1).Elemental analysis for 3 = C13H10AgF3N2O3S (MW = 439.16): found: C, 35.32; H, 2.11; and N, 6.29%; calc.: C 35.55; H, 2.30; and N, 6.38%. 1H NMR : δ = 7.57 , 7.65 , 8.61  ppm. 13C NMR : δ = 121.34 (C3 and C5), 130.66 (CH), 143.51 (C4), 150.23 (C2 and C6) ppm. IR : 3061 w, 2921 w (ν(C–H)), 1610 s, 1560 m, 1505 m, 1434 m (ν(C=C) and ν(Car = N)), 1280vs, 1251vs (νas(SO3)), 1222 s (νs(CF3)), 1156vs (νas(CF3)), 1025vs (νs(SO3)), 837 m, 553 m (γ(Car–H)). UV-Vis : 303 (ε = 4.9·104 M−1·cm−1) and 313 .Elemental analysis for 1H NMR : δ = 2.94 , 7.25 , 8.44  ppm. 13C NMR : δ = 34.56 (CH2), 123.86 (C3 and C5), 149.39 (C2 and C6), 149.64 (C4) ppm. IR : 3030 m, 2947 m, 2926 m, 2859 m, (ν(Car–H)) and (ν(C–H)), 1595 s, 1558 m, 1493 w, 1455 m, 1414 s (ν(Car = Car) and ν(Car = N)), 828vs, 807 m, 546vs, 516 m (γ(Car–H)). UV-Vis : 258 (ε = 4.4·103 M−1·cm−1).bpa data given for comparative purposes: MW = 184.24. 1H NMR : δ = 7.53 , 7.60 , 8.60  ppm. 13C NMR : δ = 121.23 (C3 and C5), 130.56 (CH), 143.32 (C4), 150.14 (C2 and C6) ppm. IR : 3101 w, 3021 m, 2985 w, 2890 w (ν(C–H)), 1595vs, 1552 m, 1496 m, 1410 s (ν(C=C) and ν(Car = N)), 835 m, 820 s, 552 m, 535 m (γ(Car–H)). UV-Vis : 303 (ε = 2.1·104 M−1·cm−1) and 315 (ε = 1.9·104 M−1·cm−1).bpe data given for comparative purposes: MW = 182.22. 1–3 were prepared fresh in DMSO (50 mg/mL) and kept at 4°C prior to use. Susceptibility testing of Candida spp.  was performed according to CLSI broth microdilution guidelines , in RPMI 1640 medium  containing 2% glucose (w/v). The highest tested concentration was 500 μg/mL, and the inocula were 1·\105 colony forming units (cfu)/mL.Stock solutions of bpa, bpe, and silver(I) complexes Pseudomonas aeruginosa PA01, Staphylococcus aureus ATCC 43300, Salmonella enteritidis ATCC 13075, Salmonella pullorum ATCC 13036, Escherichia coli NCTC 9001, Listeria monocytogenes NCTC 11994, and Enterococcus faecalis ATCC 29212 were determined in Luria-Bertani broth  in accordance with the standard broth microdilution assay for bacteria that grow aerobically, as recommended by the CLSI . The highest tested concentration was 500 μg/mL, and the inocula were 1·106 cfu/mL. MIC values were read after 24 h of incubation at 37°C as the lowest concentration to exhibit absence of growth.Minimal inhibitory concentration (MIC) values against C. albicans expressing red fluorescent protein was prepared in RPMI 1640 medium with 10% (v/v) fetal bovine serum (FBS) to induce hyphae formation  and the extinction coefficients of the complexes in the bound and free forms, respectively. In plots of [DNA]/(εa − εf) versus [DNA], Kb is given by the ratio of the slope (1/(εb − εf)) to the intercept (1/Kb(εb − εf)).UV-Vis spectrophotometric titrations were performed by maintaining the concentration of the silver(I) complexes equation :(1)DNAεa1–3. Each sample solution was scanned in the wavelength range 525–800 nm with an excitation wavelength of 520 nm. The Stern-Volmer constants (Ksv), which represent a measure of the binding propensity of the complexes to DNA, were calculated using the following equation /[EthBr] = 5, while increasing the concentration of the complexes equation :(2)F0F=1equation :(3)logF0C. albicans ATCC 10231. Briefly, DNA  was incubated with 40, 100, and 400 μM final concentration of silver(I) complexes 1–3 in Tris buffer, pH 8.5 in 50 μL reaction volume. After 2 h incubation at 30°C, 10 μL aliquots were taken, mixed with loading dye, and loaded on agarose gel. Control contained an appropriate volume of DMSO. DNA samples were run, 500 ng per lane, on 0.8% agarose gel with ethidium bromide (EthBr) against O'GeneRuler™ 1 kb Plus DNA Ladder (Thermo Scientific™) at 60 V for 1 h. Gels were visualized and analyzed using the Gel Doc EZ system , equipped with the Image Lab™ Software.DNA interaction assay using gel electrophoresis has also been conducted according to the previously published procedure  using hiμM) in Tris buffer solution (pH 7.4). The quenching of the emission intensity of tryptophan residues of BSA at 352 nm was monitored using the increasing concentration of the complexes 1–3 (up to 464.9 μM). Fluorescence spectra were recorded in the range 280–500 nm with an excitation wavelength of 275 nm. The corresponding binding constants of the complexes (KA) and apparent binding sites (n) were calculated as it was previously explained NO3}n (1), {[Ag(bpa)2]CF3SO3.H2O}n (2), and {[Ag(bpe)]CF3SO3}n (3) (X = NO3− and CF3SO3−) with the corresponding N-heterocycle in 2 : 1 molar ratio in ethanol at ambient temperature. They are moderately soluble in water, but after the addition of a small aliquot of DMSO, the complexes 1–3 become completely soluble. Different spectroscopic techniques including 1H and 13C NMR, IR, and UV-Vis were used for the structural elucidation of these complexes and the obtained data were in accordance with those determined by spectroscopic and crystallographic measurements for the same complexes previously n and [Ag(CF3COO)]n  totally prevented C. albicans filamentation and rescued the infected zebrafish of the lethal infection outcome /(εa − εf) versus [DNA] ](DMF)}, [Ag(tpp)2], [Ag(pHbza)(tpp)2], {[Ag(napr)(tpp)3](H2O)}, and [Ag(nim)(tpp)2],  [2] and [Ag(nim)(tptp)2] (tptp is tri(p-tolyl)phospine)  . From thomplexes . Neverthosphine) , as wellhospine) . In orde1–3, the competitive binding experiments based on the displacement of EthBr from ctDNA were performed  CF3SO3.H2O}n (2), and {[Ag(bpe)]CF3SO3}n (3), bpa = 1,2-bis(4-pyridyl)ethane and bpe = 1,2-bis(4-pyridyl)ethene, described in this study allowed their biological activity assessment. The present study confirms that the nitrogen-containing heterocycles, bpa and bpe, in which two pyridine rings are linked by alkyl and alkenyl group, respectively, are effective bridging ligands between two Ag(I) ions forming exclusively polynuclear complexes with NO3− and CF3SO3− as counteranions. The investigated complexes showed selective and considerable activity against four different Candida spp. and Gram-negative bacterium E. coli in comparison to the other tested bacterial strains, being moderately toxic on the normal human lung fibroblast cell line MRC-5. The type of bridging ligand in the investigated complexes plays an important role in determining their biological activity and affinity to DNA and BSA biomolecules. Complex 1 shows the best therapeutic potential  across the tested range of microorganisms. Also, both bpa-containing complexes 1 and 2 have a higher affinity to ctDNA in comparison to 3 with bpe linker, while in the case of BSA, the latter complex shows the strongest quenching ability. All these findings should be taken into consideration in design of novel silver(I) complexes as potential antimicrobial agents.Synthesis of three polynuclear silver(I) complexes, {[Ag(bpa)]NO