Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal β1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s−1 mM−1) were much lower than to laminarin (5920 s−1 mM−1). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites −1 and −2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite −1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from  In the carbohydrate-active enzymes database  are found in bacteria, fungi, plants, and marine organisms. These enzymes hydrolyze various β1-3-glucans such as linear β1-3-glucans, curdlan and paramylon  and scleO-α-l-rhamnosyl-β-glucosidase (EC 3.2.1.168). Fungal GH55 β-1,3-glucanases are physiologically involved in conidial maturation and germination in Aspergillus fumigatus , endo-β-1,3-glucanase (EC 3.2.1.39), and hesperidin 6-arzianum  through .1.58, en-glucose , 8, 9, b-glucose , 12, 13.Phanerochaete chrysosporium exo-β-1,3-glucosidase PcLam55A . The active site is located at the interface of the N- and C-domains. The Glu residue  in C8-L3 of the C-domain serves as a general acid catalyst with the help of the adjacent Tyr residue (Tyr-505 of SacteLam55A) in the orientation by a hydrogen bond. The proposed proton relay network of four amino acid residues  activates a water molecule, which attacks the anomeric carbon of the substrate from the other side of the scissile β-glucosidic bond . The tryptic digests of MnLam55A were subjected to LC-MS/MS analysis to identify the MnLam55A gene from the M. nivale genome (Microdochium bolleyi (NCBI ID: KXJ95237.1). Among characterized GH55 enzymes, the highest identity at 64% was found for LamAI from Trichoderma viride U-1 (B). MnLam55A possesses all of the highly conserved residues of GH55 enzymes for the formation of the catalytic site and subsite −1 as follows: the general acid catalyst, Glu-614; its adjusting residue, Tyr-617; the proton relay network-activating substrate water, Glu-140, Ser-203, Gln-172, and Glu-591; the subsite −1 forming residues, Glu-140, Trp-552, Asp-557, Glu-591, and His-592; and the aromatic block, Phe-143, Trp-552, and Trp-554   was purified to homogeneity from the culture supernatant of SDS-PAGE A. The trride U-1 B. MnLam5nLam55A) .Figure 3Komagataella pastoris transformant harboring the gene encoding MnLam55A with the N-terminal signal sequence of α-factor replacing the original signal sequence. In 1.2 l of the culture broth, 491 U of laminarinase activity was produced after 96 h of cultivation. Purified recombinant MnLam55A  showed 16.9 U/mg of specific activity and a single band of 67 kDa on SDS-PAGE, consistent with those of the native enzyme (A). The apparent molecular mass on SDS-PAGE was lower than the calculated mass (80 kDa) from amino acid sequences. This smaller apparent molecular mass on SDS-PAGE was also reported in other fungal GH55 endo-β-1,3-glucanases BGN13.1 from T. harzianum (T. viride (A and B). This enzyme retained ≥95% of the original activity after incubation in a pH range of 4.3 to 9.3  and at ≤45 °C (for 20 min) (A and B).Recombinant MnLam55A was extracellularly produced in a e enzyme A. The ap. viride , A and B 20 min) , A and Bd-glucose, gentiobiose, and gentiotriose, which were possibly released from nonreducing end of laminarin through exo-acting hydrolysis (A). This result suggests that MnLam55A has both endo- and exo-acting activities on laminarin but acts it mainly in an endo-acting manner. Using 62.5-fold higher concentration of enzyme, the reaction approached the complete digestion of laminarin (B and C). d-Glucose and oligosaccharides with degrees of polymerization (DPs) of 2 to 4 gradually accumulated with decrease of laminarin (DP ≥10). After 25.5 h of reaction, 90% of laminarin was degraded to monosaccharide and oligosaccharides of DP2–4 (B). The disaccharide, one of the complete degradation products accumulated in the reaction, migrated similarly to the gentiobiose standard in the TLC analysis. The trisaccharide and tetrasaccharide were isolated by gel-filtration column chromatography, and their chemical structures were analyzed by electrospray ionization (ESI)-mass spectrometry (MS) and NMR. In MS spectra of the trisaccharide and the tetrasaccharide, [M+Na]+ ion peaks of m/z 527.16 and 689.21 were detected, respectively. From the NMR analysis, the chemical shifts from 13C NMR spectra of the trisaccharide and the tetrasaccharide matched those of gentiotriose (d-glucose and gentiooligosaccharides (DP2–4) through its exo-acting activity.The reaction products from laminarin were monitored. At the early stage of the reaction, total molar concentration of reducing sugars was 4-fold higher than the sum of those of drolysis A. This raminarin , B and Cof DP2–4 B. The diiotriose  and gentiotriose , respectd-glucosyl residues: a, Glc1→6; b, →3Glc1→6; c, →6Glc1→3; and d, →3Glc1→3 . In addition to these peaks, two correlation peaks derived from the newly produced residues (α and β) were observed in the COSY 2D-NMR spectrum of the 15-min reaction product . The heteronuclear multiple bond correlation 2D-NMR spectra of this reaction product showed the clear correlation peak of 1-C from a and b and 6-H from α and β (δH 4.19→δC 103.4), although those of 1-H from a and b and 6-C from sugar residues α and β (δH 4.50→δC 69.4) were covered by other peaks . The correlation peaks of 3-C from α and β and proton signals from any other d-glucosyl residues were not observed . These results indicated that α and β were 6-O-β-glycosylated d-glucose residues at the reducing end (→6Glc).In order to determine the initial cleavage site of laminarin, NMR analysis of early reaction products from laminarin was performed. COSY 2D-NMR spectrum of laminarin showed four correlation peaks between 1-H and 2-H, which corresponded to the four D). The 1-H signals of a and b were obtained by 1H NMR analysis at 27 °C, while those of c and d were conducted at 60 °C to avoid overlapping the large signal of H2O . Anomeric proton signals of sugar residues, a–d, are shown as a1–d1, respectively, and 1-H signals of the reducing end residue of the reaction products are shown as α1 and β1. The ratio of c1 decreased the most, and it was followed by d1 and b1 (D). The signal of a1 slightly increased and that of α1+β1 significantly increased. The sum of the decreasing ratio of b1 and d1 was almost equal to that of c1, and the increasing ratio of a1 was almost equal to the decreasing ratio of b1. These results indicated that MnLam55A cleaved the β1-3-linkages between sugar residues c and b/d (nonreducing end 6-O-glycosylated β1-3-glucosidic linkage of β1-3-linked laminarioligosaccharide portions of laminarin) and produced the oligosaccharides harboring 6-O-β-glycosylated d-glucose residues at reducing end (α and β) (G).Differences of the integration ratio to the sum of the integrations of each 1-H signal between 0- and 15-min reaction solutions were calculated D. The 1-1 and b1 D. The siα and β) G.A). At the early stage of the reactions with laminarioligosaccharides (DP4–6), d-glucose was most commonly produced (B–D), indicating the dominant exo-type hydrolysis of laminarioligosaccharides. To identify the terminal of the substrates releasing d-glucose, MS analysis of the products from laminaripentaose hydrolyzed in H2O and H218O was performed. A mass peak of m/z 181.06, corresponding to [M−H]− of 18O-containing d-glucose, was observed only in the spectrum of the reaction with H218O (E and F). In contrast, the mass peak corresponding to [M−H]− of 18O-containing laminaritetraose was not observed. The weak mass signal at m/z 667.22, two units higher than that of laminaritetraose, was observed in both the reactions with H2O and H218O and was considered to be the natural isotope of laminaritetraose (G and H).Reactions of MnLam55A with laminarioligosaccharides (DP2–6) were analyzed. MnLam55A degraded laminaritriose and longer laminarioligosaccharides, but not laminaribiose A. At theproduced , B–D, inth H218O , E and Ftetraose , G and H−1), but not on scleroglucan , curdlan (liner β1-3-glucan), paramylon (liner β1-3-glucan), and barley β1-3/1-4-glucan (<0.110 s−1). MnLam55A showed 1400-fold higher kcat/Km to laminarin (5920 s−1 mM−1) than even to laminarioligosaccharides of DP 3, which showed the highest kcat/Km (4.21 s−1 mM−1) among the laminarioligosaccharides (Reaction velocities for various β-glucans and laminarioligosaccharides (DP2–6) were measured. MnLam55A acted on laminarin . RMSD for Cα atoms of MnLam55A and GH55 exo-β-1,3-glucosidases is 1.38 Å to PcLam55A (PDB ID: 3EQO), 1.25 Å to CtLam55 (PDB ID: 5M60), and 2.79 Å to SacteLam55A (PDB ID: 4PEW). Thus, the whole structure of MnLam55A is more similar to the fungal enzymes than to the bacterial one. The active site and substrate-binding site of MnLam55A were compared with those of PcLam55A (PDB ID: 3EQO) and SacteLam55A (PDB ID: 4TZ1) . In MnLam55A, the catalytic site and subsite −1 are formed by Glu-140, Gln-172, Ser-203, Trp-552, Asp-557, Glu-591, His-592, Glu-614, and Tyr-617, the aromatic block is formed by Phe-143, Trp-552, and Trp-554, and subsite +1 is formed by Gln-225, Tyr-617, Phe-677, and Phe-678. These residues are spatially conserved well with those of exo-β-1,3-glucosidases . Residues forming subsite +2 of MnLam55A are Gln-225 and Trp-738, which are spatially placed at similar positions of the corresponding residues of exo-β-1,3-glucosidases, although their orientation is less conserved compared to the residues at subsites −1 and +1 . As the plus subsites for the binding of longer oligosaccharides, the aromatic residues suggested in SacteLam55A (Trp-196 at subsite +2 and Tyr-194 at subsite +5) and those on the putative substrate binding groove in PcLam55A  are not observed in MnLam55A. Docking simulation of MnLam55A with 6Ⅲ-O-glucosyl laminaritriose was performed to predict a binding mode of the nonreducing end β1-6-linked GlcⅣ group to the enzyme. In the docked model, GlcⅢ of the ligand was accommodated at subsite −1, where the nonreducing end d-glucosyl residue of laminaritriose was predicted as shown in Figure 7B . The GlcⅣ residue was accommodated in possible subsite −2, corresponding to the expected space in the fungal GH55 exo-β-1,3-glucosidases (Ⅳ in subsite −2 (D and E). In the structure of PcLam55A (PDB ID: 3EQO), the corresponding His-576 was similarly located but more residues seemed to be involved in the formation of the pocket-shaped subsite −2 (F–H). In contrast, MnLam55A possesses no equivalent residues, particularly to Ser-583, mainly due to the differentially oriented C6-L3. The 3-hydroxy group of GlcⅣ in the MnLam55A structure was located in an open space on an extended cleft structure (C and D). This cleft is formed at the interface of the two domains: C6-L3 in the C-domain  to one side, and C4-L1 and C2-L1 of the N-domain involving Leu-141, Arg-138, Thr-135, Glu-74, and Trp-75 for the other . The extended cleft was expected to form the minus subsites beyond subsite −2.The crystal structure of MnLam55A was determined in unliganded form at 2.4 Å resolution. The solved structure contains two molecules of MnLam55A in the asymmetric unit. The entire structure consists of two β-helical domains of 3-solenoid connected by a linker  and exhibited endo-type action in the reaction with laminarin (A) but no activity with β1-3-glucans containing no β1-6-linkages in the main chain, whereas typical endo-β-1,3-glucanases in families other than GH55 randomly degrade the β1-3-glucans (G). As this enzyme almost completely degraded laminarin to d-glucose and gentiooligosaccharides (O-β-d-glucosyl gentiooligosaccharides after complete degradation of laminarin (C) suggested that MnLam55A hydrolyzes the last reducing-end β1-3-d-glucosidic linkage when linked to gentiooligosaccharides, although laminaribiose is not a substrate of this enzyme. MnLam55A acted similarly on nonreducing terminal β1-3-d-glucosidic linkages of laminarioligosaccharides and laminarioligosaccharide moieties of laminarin but with very different kcat/Km values . The exo-type action of MnLam55A on laminarioligosaccharides and laminarioligosaccharide moieties in the main chain of laminarin is attributed to the structure for exo-acting enzymes. The possible subsite −2 structure of MnLam55A was probed by the docking study with 6Ⅲ-O-d-glucosyl laminaritriose . The space accommodating the nonreducing end d-glucosyl residue (GlcⅣ) was equivalent to the pocket structure in fungal GH55 exo-β-1,3-glucosidases for binding of the β1-6-branching moiety of substrate (C–H). PcLam55A, however, additionally has Asn-147, Ser-583, and Gln-585 involved in the formation of the closed pocket structure, preventing 3-O-glycosylated d-glucosyl group from binding to subsite −2 (F–H). Asn-147 and Gln-585 are highly conserved in exo-type enzymes, but endo-type enzymes including MnLam55A share no corresponding residues . Thus, this cleft possibly forms minus subsites beyond subsite −2 to accommodate the laminarin main chain linked to 3-O of the d-glucosyl residue bound to subsite −2. The cleft-forming residues of N-domain, Trp-75, Thr-135, and Leu-141 of MnLam55A, are conserved well in other endo-β-1,3-glucanase sequences, which suggests their importance for the formation of minus subsites. Another structure directly involved in the formation of this cleft in MnLam55A is C6-L3 of the C-domain, whereas the corresponding loop of GH55 exo-β-1,3-glucosidases are differentially located close to subsite −2 to form the closed pocket-shaped structure (C–H). The difference in the C6-L3 localization is presumably caused by the structural difference of the adjacent C5-L3 (C–H). C5-L3 of MnLam55A is shorter than the corresponding loops of fungal exo-type enzymes  (7B). Laminaritriose is probably required for productive binding. In contrast, some aromatic residues such as Trp-196 and Tyr-194 in SacteLam55A at subsites +2 and +5, respectively  , 14, 15.A)  and 7B. ectively  are invoIn conclusion, we found that endo-acting hydrolysis of MnLam55A cleaves the β1-3-linkages at the nonreducing end of the laminarioligosaccharide moiety adjacent to β1-6-linkages in the laminarin main chain with significantly higher affinity than exo-wise hydrolysis of laminarioligosaccharides. Crystal structure determination and the docking analysis revealed that MnLam55A shares with fungal GH55 exo-β-1,3-glucosidases overall structures and subsites −2 to +2, including the binding mode of the gentiobiose moiety in subsites −1 and −2, but the possible substrate-binding cleft extending from subsite −2 was found in MnLam55A structure for the first time. The relevant structures are similar in other GH55 endo-β-1,3-glucanases. Based on these findings, we present the substrate binding modes in GH55 endo-acting β-1,3-glucanases .M. nivale strain MCW222-7 was originally obtained from wheat in Memuro, Hokkaido, Japan and stored at −20 °C. It was grown on potato dextrose agar medium  and cultured in potato dextrose broth (0.8 l) following previous work , was loaded on a DEAE Sepharose Fast Flow column  equilibrated with 10 mM sodium phosphate buffer (pH 7.0). Nonadsorbed protein, eluted by the same buffer, was separated on a Butyl Sepharose Fast Flow 4 column  equilibrated with 10 mM sodium phosphate buffer (pH 7.0) containing 1.2 M ammonium sulfate. After washing the column with the same buffer, adsorbed protein was eluted with a linear gradient of 1.2 to 0 M ammonium sulfate. Active fractions were concentrated by ultrafiltration using Amicon Ultra YM30 , loaded on a Sephacryl S-200 column , and equilibrated with 10 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl. Active fractions were collected, and 2.4 μg of the purified enzyme was further separated by SDS-PAGE. The single-protein band of MnLam55A was cut out and in-gel tryptic digest was performed using In-Gel Tryptic Digestion kit (Thermo Fisher Scientific). The resulting peptides, purified by the Pierce C18 Spin column (Thermo Fisher Scientific), were analyzed with LC-MS/MS Paradigm MS2 (Michrom BioResources) following previous research  with a draft genome sequence of M. nivale  using In-Fusion HD Cloning Kit (Takara Bio). The cloned DNA was sequenced using an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). This SacⅠ-linearized plasmid was introduced into K. pastoris X-33 by electroporation. The transformant was cultured following published research  was measured. A reaction mixture (100 μl) consisting of 4 mg/ml laminarin, 40 mM sodium acetate buffer (pH 5.1), 0.2 mg/ml bovine serum albumin (BSA), and appropriate concentration of enzyme was incubated at 30 °C for 12 min. Reaction mixture (10 μl) was taken every 3 min, and reducing sugar was quantified by measuring A560 according to the copper-bicinchoninate method  titrated with 0.5 M NaOH) was used. The stable pH range was determined by residual activity after incubation of 348 nM MnLam55A in 20 mM Britton–Robinson buffer (pH 2.5–12.0) at 4 °C for 24 h. The optimum temperature was determined by the reaction rates in the standard assay but 10 to 70 °C. The stable range of MnLam55A against temperature was determined from residual activity after incubation of 174 nM MnLam55A in 67 mM sodium acetate buffer (pH 5.1) at 30 to 65 °C for 20 min. The range in which the enzyme retained ≥95% of the original activity was regarded as stable range. Three independent replications of each experiment were conducted.2O. 1H NMR, COSY 2D-NMR, 13C NMR, heteronuclear single quantum coherence 2D-NMR, and heteronuclear multiple bond correlation 2D-NMR spectra were measured using Avance Neo (Bruker). Data were analyzed using TopSpin version 3.6.2 .To analyze the initial products, the reaction with laminarin was performed using a low concentration of the enzyme for a short period. A reaction mixture consisting of 4 mg/ml laminarin, 40 mM sodium acetate buffer (pH 5.6), 0.2 mg/ml BSA, and 13.9 nM MnLam55A, was incubated at 30 °C for 15 min. The reaction was terminated by heating at 100 °C for 3 min. Reducing sugar was quantified as described above, and sugar content was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection under following conditions: column, CarboPac PA-1 ; elution, linear gradient of 0 to 250 mM sodium acetate in 0.2 M NaOH; flow rate, 0.8 ml/min; and detection, pulsed amperometry. For NMR analysis, the reaction solutions (740 μl) at 0 and 15 min were lyophilized and dissolved in D254 plate (Merck) developed with 2-propanol:1-butanol:water = 12:3:4 (v/v/v) and HPLC using Aminex HPX-42A Carbohydrate column  with water at 0.5 ml/min, and a refractive index detector, RI-2031 Plus (Jasco).The progress of the reactions was monitored as follows. A reaction mixture of 10 mg/ml laminarin, 11 mM sodium acetate buffer (pH 5.6), 0.05 mg/ml BSA, and 870 nM MnLam55A was incubated at 30 °C for 25.5 h. Aliquots (100 μl) taken at several timepoints were heated at 100 °C for 3 min. Sugar content of the samples was analyzed by TLC using a Silica gel 60 FFrom the reaction products, the trisaccharide and tetrasaccharide products were prepared. A reaction (10 ml) was made as above but with 174 nM MnLam55A for 24 h. The trisaccharide and tetrasaccharide were separated by gel-filtration column chromatography using Toyopearl HW-40s  with water under 1.8 ml/min and analyzed by ESI-MS using Exactive Plus (Thermo Fisher Scientific) and NMR as described above.A reaction mixture (50 μl) of 10 mM laminarioligosaccharides , 10 mM sodium acetate buffer (pH 5.6), 0.2 mg/ml BSA, and 17.4 nM MnLam55A was incubated at 30 °C for 180 min. Aliquots (10 μl) taken at several timepoints were heated at 100 °C for 3 min. The samples were analyzed by TLC as described above. Progress of initial reactions was monitored by analyzing samples  with high-performance anion-exchange chromatography with pulsed amperometric detection.2O and H218O was performed as follows. The reaction was made in a mixture (25 μl) of 2 mM laminaripentaose, 40 mM sodium acetate buffer (pH 5.6), 0.2 mg/ml BSA, and 139 nM MnLam55A in H2O or 99.2% (v/v) H218O (Sigma) at 30 °C for 30 min. The samples were heated at 100 °C for 3 min, dried, and analyzed by ESI-MS as described above.MS analysis of laminaripentaose-hydrolyzed products in Hd-glucose was quantified with a Glucose CⅡ Test  . Three independent replications of each experiment were conducted.Reaction velocities with β-glucan were measured with the following substrates: laminarin, scleroglucan (Biosynth), curdlan , paramylon (Sigma), and barley β1-3/1-4-glucan (Sigma). The reactions were made in a mixture (100 μl) of 1 mg/ml β-glucan, 40 mM sodium acetate buffer (pH 5.6), 0.2 mg/ml BSA, and MnLam55A (1.86 nM for laminarin and 186 nM for the others) at 30 °C for 15 min. The reaction for the kinetic analysis with laminarin was done in a reaction mixture (100 μl) of 0.2 to 2 mg/ml (6.4–64 μM) laminarin, 40 mM sodium acetate buffer (pH 5.6), 0.2 mg/ml BSA, and 3.48 nM MnLam55A, at 30 °C for 12 min. Reducing sugar was measured as described above. For laminarioligosaccharides (DP3–6), reactions were similar to those for laminarin but containing 2 to 10 mM laminarioligosaccharides and 34.8 nM MnLam55A, at 30 °C for 10 min. Release of hemical) , 26 aftehttps://xds.mr.mpg.de/)  was prepared by mixing 5.1 mg/ml MnLam55A in 10 mM Hepes-NaOH buffer (pH 7), and 10 mM glucose with the same volume of reservoir solution consisting of 0.1 M Hepes-NaOH buffer (pH 7), 1 M disodium succinate, and 10 mg/ml polyethylene glycol monomethyl ether 2000. Crystallization was observed within 37 days at 20 °C. Crystals were removed from the crystallization solution and flash-cooled. Diffraction data were collected on a beamline BL45XU at SPring-8. The datasets were indexed, integrated, scaled, and merged using the XDS program suite (mpg.de/) . The asympg.de/)  were 3.0https://phenix-online.org/) (Fo − Fc and Fo − Fc electron densities using COOT (https://www.ccp4.ac.uk/) . All structure figures were generated by PyMOL ver. 2.6.0a0 .The structure of MnLam55A was determined by the molecular replacement method with the program AutoMR in the PHENIX program package (ne.org/) , 30. Thene.org/)  was used.ac.uk/) , 32. Wat.ac.uk/)  are showⅢ-O-glucosyl laminaritriose was constructed by BIOVIA Discovery Studio Visualizer (Dassault Systèmes BIOVIA). In the simulation, water and glycerol molecules in the structure of MnLam55 A were removed. The 6Ⅲ-O-glucosyl laminaritriose was docked into the active site of MnLam55A using the grid box with a spacing of 1 Å and dimension of 40 × 40 × 114, centered at positions of 0 (x), 0 (y), and 0 (z).A docking simulation was performed by AutoDock Vina (ADT version 1.5.6)  to estimThe data underlying this article are available upon reasonable request to the corresponding author.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Hibiscus cannabinus seed (PEHc).Reports have implicated diabetes mellitus (DM) and Alzheimer’s disease (AD) as some of the global persistent health challenges with no lasting solutions, despite of significant inputs of modern-day pharmaceutical firms. This study therefore, aimed to appraise the in vitro antioxidant potential, enzymes inhibitory activities, and as well carry out in silico study on bioactive compounds from polyphenolic-rich extract of https://www.rcsb.org/).In vitro antioxidant assays were performed on PEHc using standard methods while the identification of phytoconstituents was carried out with high performance liquid chromatography (HPLC). For the in silico molecular docking using Schrodinger’s Grid-based ligand docking with energetics software, seven target proteins were retrieved from the database (p < 0.05) inhibition of ABTS (IC50 = 218.30 ± 0.87 µg/ml) and 1, 1-diphenyl-2-picrylhydrazyl free radicals (IC50 = 227.79 ± 0.74 µg/ml). In a similar manner, the extract demonstrated a significant (p < 0.05) inhibitory activity against α-amylase (IC50 = 256.88 ± 6.15 µg/ml) and α-glucosidase (IC50 = 183.19 ± 0.23 µg/ml) as well as acetylcholinesterase (IC50 = 262.95 ± 1.47 µg/ml) and butyrylcholinesterase (IC50 = 189.97 ± 0.82 µg/ml), respectively. Furthermore, In silico study showed that hibiscetin (a lead) revealed a very strong binding affinity energies for DPP-4, (PDB ID: 1RWQ) and α-amylase (PDB ID: 1SMD), gamma-tocopherol , cianidanol for α-glucosidase; PDB ID: 7KBJ and kaempferol for Poly [ADP-ribose] polymerase 1 (PARP-1); PDB ID: 6BHV, respectively. More so, ADMET scores revealed drug-like potentials of the lead compounds identified in PEHc.HPLC technique identified twelve chemical compounds in PEHc, while antioxidant quantification revealed higher total phenolic contents (243.5 ± 0.71 mg GAE/g) than total flavonoid contents (54.06 ± 0.09 mg QE/g) with a significant (As a result, the findings of this study point to potential drug-able compounds in PEHc that could be useful for the management of DM and AD.The online version contains supplementary material available at 10.1186/s12906-023-04023-7. The importance of bioactive plant secondary metabolites in the management of a variety of human diseases, including cancer, diabetes mellitus (DM), cognitive dysfunction or memory impairment, such as Alzheimer’s disease (AD), Parkinson’s disease, and others, has recently been highlighted , 2. DM aDM is a chronic metabolic disease that is one of the most serious public health issues of the twenty-first century . It is aDM is classified into two types: type 1 (T1DM) and type 2 (T2DM) . T2DM isSimilarly, AD is a progressive neurodegenerative brain disorder –24. It iNowadays, there are more and more new therapeutic targets available for drug discovery as a result of the completion of the human genome project . These dHibiscus cannabinus (H. cannabinus) seed, commonly known as kenaf, is an annual herbaceous crop, which belongs to the Malvaceae family that probably originated from sub-Saharan Africa  polymerase 1 (PARP-1) (PDB ID: 6BHV), acetylcholinesterase (AChE)-(PDB ID: 4EY7), and butyrylcholinesterase (BChE)-(PDB ID: 7B04) were obtained from protein data bank  and standard drugs for individual targets (obtained from medexpress and drugbank) were prepared using the LigPrep 2.4 software . The OPLTo evaluate the docking parameters, all drugs were docked into the DPP-4, (PDB ID: 1RWQ), α-amylase (PDB ID: 1SMD), glucagon-like peptide-1 receptor (PDB ID: 3C59), α-glucosidase (PDB ID: 7KBJ), poly [ADP-ribose] polymerase 1 (PARP-1) (PDB ID: 6BHV), acetylcholinesterase (AChE)-(PDB ID: 4EY7), and butyrylcholinesterase (BChE)-(PDB ID: 7B04) proteins retrieved from the protein database, using Schrodinger’s Grid-Based Ligand Docking with Energetics (GLIDE) software. In the receptor grid generation with a partial atomic charge of 0.25, the scaling factor for protein van der Waals radii was 1.0 to soften the potential for nonpolar regions of the receptors. The GLIDE docking parameters were set to the flexible potential function’s default. For all docking studies, no limitations or constraints were applied. GLIDE 5.6’s receptor grid generation module was used to define the active site for docking ligands. Co-crystallied ligands were chosen, and grids were generated around the active site of 1RWQ, 1SMD, 3C59, 7KBJ, 6BHV, 4EY7 and 7B04 with receptors van der Waals scale of 0.9 for non-polar atoms. The active sites were determined by a radius of 10 around the crystal structure’s ligands , 62.binding), was computed using the method of Lyne et al.  polymerase 1 (6BHV). Table S6. Post docking SP, XPG and MM-GBSA scores of compounds from polyphenolic-rich extract of Hibiscus cannabinus and a standard (Rivastigmine) with Butylcholinesterase (7B04). Table S7. Post docking SP, XPG and MM-GBSA scores of compounds from polyphenolic-rich extract of Hibiscus cannabinus and a standard (Rivastigmine) with Acetylcholinestrase (4EY7).


The purinergic receptor P2X7 plays a crucial role in infection, inflammation, and cell death. It is thought that P2X7 receptor stimulation triggers processing and release of the pro-inflammatory cytokine interleukin (IL)-1β by activation of the NLRP3 inflammasome; however, the underlying mechanisms remain poorly understood.Modulation of IL-1β secretion was studied in THP-1 macrophages. Adenosine 5’-triphosphate (ATP), BzATP, nigericin and pharmacological inhibitors of P2X receptors, inflammatory caspases and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome were used to characterize signaling.+ as well as inhibition of caspase-1 or serine proteases maintained IL-1β release in macrophages stimulated with P2X7 receptor agonists at 50%.In primed macrophages, IL-1β release was increased after P2X7 receptor activation by ATP and 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP). Pharmacological inhibition or genetic knockout of NLRP3 does not completely inhibit IL-1β release in TLR2/1-primed macrophages. Increase in extracellular KOur findings suggest a previously unrecognized mechanism of P2X7 receptor mediated IL-1β release and highlight the existence of an NLRP3-independent pathway in human macrophages.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01356-1. IL-1β+ efflux , 12. How+ efflux and activation of the NLRP3 inflammasome [ATP is one of the best studied DAMPs , 14. Extammasome .+ efflux and requires activation of caspase-1 and serine proteases, while the other mechanism is independent of K+ efflux, caspases, as well as serine, cysteine, and aspartic proteases.To date, inflammatory signaling has often been studied in mouse bone marrow-derived macrophages , however5 cells/ml. Cells were used from passage 4 to 25 and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cell lines were regularly tested negative for mycoplasma contamination . For generating THP-1 derived macrophages, THP-1 monocytes were seeded into 24-well plates at a density of 4 × 105cells/ml in growth medium including 25 ng/ml PMA . After 48 h, adherent cells were carefully washed with PBS  and rested in PMA-free medium for 24 h. PBMCs were isolated from buffy-coat donations  by density gradient centrifugation using Bicoll separation media . HEK-blue IL-1β cells were cultured in Dulbecco's modified Eagle's medium  containing 4.5 g/l glucose, 10% (v/v) heat-inactivated fetal bovine serum , 2 mM l-glutamine (G7513), 100 U/ml penicillin, 100 μg/ml streptomycin , 100 μg/ml normocin, 100 μg/ml zeocin . Cells were used from passage 3 to 20 and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. To determine if THP-1 stimulated cells secrete bioactive form of IL-1β a total of 50 μl of THP-1 supernatant was transferred to a 96-well tissue culture–treated plate and mixed with 5 × 104 IL-1β reporter cells in test media (culture media without normocin and zeocin) and incubated for 20 h at 37 °C. After IL-1β receptor stimulation of HEK-Blue cells, NF-κB and AP-1 activation-induced production of secreted embryonic alkaline phosphatase (SEAP) that can be determined with the colorimetric substrate QuantiBlue  by reading the optical density (OD) at 620 nm.THP-1 cells  or THP-1 KO NLRP3 cells  were cultured in RPMI 1640  containing 100 U/ml penicillin, 100 μg/ml streptomycin (P4333), 2 mM L-glutamine  and 10% heat-inactivated fetal bovine serum  at a density of 2–8 × 10Escherichia coli 0111:B4 (tlrl-3pelps), Pam2CSK4 (tlrl-pm2s-1), Pam3CSK4  or growth medium for 3 h. Afterwards PBMCs and THP-1 macrophages were stimulated for additional 3 h or THP-1 monocytes for 6 h with 300 μM BzATP (2'/3'-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, NU-1620–25), 5 mM ATP  10 μM nigericin . In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 , irreversible P2X7 receptor antagonist oxidized ATP , P2X4 receptor antagonist 5-BDBD , P2X receptor antagonist PPADS , NLRP3 inhibitor MCC950  or Bay 11–7082 , Caspase-1 inhibitor Ac-YVAD-cmk , pan-caspase inhibitor Z-VAD-fmk , serine protease inhibitor AEBSF , cysteine protease inhibitor E64 , aspartic protease inhibitor pepstatin A  1 h before stimulation. To determine the optimal concentration of caspase inhibitors required to inhibit IL-1β secretion, we incubated increasing concentrations of Ac-YVAD-cmk and Z-VAD-fmk together with nigericin . To determine the contribution of the NLRP3 inflammasome, THP1 KO NLRP3 macrophages were primed with 1 μg/ml Pam3CSK4 for 3 h followed by stimulation with BzATP, ATP or nigericin for 3 h.PBMCs as well as THP-1 monocytes and differentiated THP-1 derived macrophages were preincubated with LPS from 3CSK4-primed PBMCs or THP-1 macrophages or after 6 h of stimulation of Pam3CSK4-primed THP-1 monocytes cell culture supernatants were collected and analyzed for IL-1β release using commercially available ELISA kits .After 3 h of stimulation of PamLDH assay was performed according to the manufacturer’s instructions . The percentage of LDH release was calculated compared to 100% cell lysis control.Total RNA isolation was performed using innuPREP RNA Mini Kit 2.0  according to the manufacturer’s protocol. Hence, cDNA was synthesized with the help of iScript cDNA Synthesis Kit . Quantitative real-time RT-PCR (qRT-PCR) was performed as previously described . PrimersFold difference in gene expression was normalized to the housekeeping gene GAPDH showing the most constant expression levels. The reaction mix containing cDNA template, primers and SYBR Green  was run under the conditions as previously described.P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. For studies of inhibitory effects ATP, BzATP or nigericin induced IL-1β release was set to 100%. All other values were calculated accordingly. Statistical differences were assessed by one-sample t test against 100%. Statistical analysis was performed using GraphPad Prism software.Data are expressed as means + SEM. For multiple comparisons, statistically significant differences were determined by one-way ANOVA followed by a Dunnett´s or Tukey´s post-test and considered significant at *3CSK4 followed by stimulation with ATP, BzATP, and nigericin. We confirmed that priming was necessary to induce IL-1β release in PBMCs  with the TLR2/1 ligand Pam3CSK4-primed THP-1 macrophages stimulated with ATP and BzATP resulted in a significant increase in absorbance confirming that ATP and BzATP induced the bioactive form of IL-1β. Although nigericin strongly induced IL-1β release in THP-1 macrophages we did not observe a significant increase in absorbance compared to control, which might be explained by cytotoxic effects of remaining nigericin in the supernatant.To investigate the bioactivity of the secreted IL-1β, we added the supernatants of stimulated THP-1 macrophages to HEK-blue IL-1β reporter cells , neutrophil elastase (ELANE), or cathepsin G (CTSG), which have been shown to alternatively process IL-1β compared to caspase-1 [To elucidate the involvement of other proteases in IL-1β secretion, we used inhibitors of serine-, cysteine-, and aspartic proteases. In the presence of the serine protease inhibitor AEBSF we observed a decrease in IL-1β secretion mediated by ATP, BzATP and nigericin Fig. A. When Aaspase-1 , were no2CSK4 and the TLR4 ligand LPS and observed increased IL-1β release after stimulation with ATP, BzATP, or nigericin (Fig. IL1B and IL18 did not show a significant difference between TLR2 and TLR4 signaling (Fig. 3CSK4. In contrast, inhibition of NLRP3 reduced BzATP-induced IL-1β cytokine levels to 44.6% and 57.0% in LPS- and Pam2CSK4-primed cells, respectively, indicating that TLR signaling differentially modulates signal transduction of P2X7-mediated IL-1β release (Fig. Not only TLR2/1 ligands can serve as priming signal for NLRP3 inflammasome activation. Therefore, we also used the TLR2/6 ligand Pam3CSK4-primed human THP-1 macrophages lacking NLRP3, stimulation with ATP led to a significant increase in IL-1β release. These effects were even more pronounced for BzATP, a synthetic ATP derivative and a more potent P2X7 receptor ligand [Although our findings support the importance of the NLRP3 inflammasome in P2X7 receptor-mediated IL-1β release , we propr ligand .A limitation of the present study is the use of the THP-1 cell line, which may deviate from primary cells. THP-1 cells are widely used to study the NLRP3 inflammasome , howeverWe confirmed full NLRP3-dependency of nigericin induced IL-1β release , however+ efflux, which differs from the previous concept established in mouse macrophages [High extracellular potassium concentrations inhibit NLRP3 activation and IL-1β release , 12. Ourrophages . Our finrophages , althougrophages , but fur+ efflux is associated with caspase-1 for P2X7 receptor-dependent IL-1β release.Caspase-1 is a central player in inflammasome-induced IL-1β release . AlthougCaspase and NLRP3 independent mechanisms for IL-1β processing have been described, including serine proteases that are involved in caspase-1-independent IL-1β release , 44. A s3CSK4 led to completely NLRP3-independent IL-1β release in BzATP-stimulated macrophages, while priming with TLR2/6 and TLR4 ligands led to some degree of NLRP3 dependency. Therefore, it would be interesting to determine the underlying mechanisms in the NLRP3 priming pathway triggered by TLR ligands.Lastly, priming with TLR agonists differentially modulates NLRP3 dependency of P2X7 mediated IL-1β release. Priming with the TLR2/1 ligand PamIn summary, we propose an independent pathway of NLRP3 and caspase for IL-1β release following activation of the P2X7 receptor in human macrophages Fig. . In thisAdditional file 1: Supplementary figures S1-S3.


Introduction: Dysfunction of the endothelium impairs its’ protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses.Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells.Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells.Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs. Endothelial dysfunction, a hallmark of inflammation, is linked to the pathogenesis of vascular diseases and results in barrier disruption, inflammation and apoptosis . There ain vitro and in vivo -41 site within the N-terminus . However in vivo . Howeverβ-arrestin interaction with activated GPCRs is mediated by receptor phosphorylation and engagement with the transmembrane core . G proteIn the present study, we examined the mechanisms by which APC modulates tumor necrosis factor-α (TNF-α) cytokine induced inflammatory responses in human cultured endothelial cells. We show that APC stimulation alters phosphorylation of several TNF-α signal pathway components and attenuates of TNF-α induced vascular cell adhesion molecule-1 (VCAM-1) expression independent of mRNA stability. Unexpectedly, we found a role for S1PR1 and GRK2 in APC/PAR1 mediated suppression of cytokine-induced VCAM-1 expression. This study reveals that different APC/PAR1 cytoprotective responses are mediated by discrete β-arrestin-2-driven signaling pathways modulated by specific co-receptors and GRKs.2 in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) and 20% preconditioned media as we previously described  for 1 h prior to agonist, antagonist and/or inhibitor treatments at 37°C.HUVEC-derived endothelial EA. hy926 cells  were grown at 37°C in 8% COescribed . EndotheEndothelial EA. hy926 cells were pretreated with or without 20 nM APC for 3 h at 37°C . Cells were then stimulated with 10 ng/mL TNF-α  for the indicated times at 37°C and responses measured. To block PAR1 signaling, serum-starved cells were pretreated at 37°C with 10 µM Vorapaxar  for 1 h before APC treatment. S1PR1 signaling was inhibited by incubation with 10 µM W146  for 30 min at 37°C. GRK2 was inhibited by preincubating endothelial cells with 50 µM Compound (CPMD)101  at 37°C for 1 h in serum-free media before APC stimulation., anti-phospho-p38 T180/Y182 (#4511) and anti-p38 MAPK (#8690) antibodies , anti-β-actin , anti-GRK2/3 , anti-GRK4-6  and anti-GAPDH . Secondary antibodies were anti-mouse or anti-rabbit HRP conjugated antibodies .The following antibodies were used in this study: anti-VCAM-1 (#13662S), anti-ICAM-1 (#4915S)https://endotheliomics.shinyapps.io/endodb/) . The relative mRNA levels, including replicates, of each of the 5 independent studies were analyzed by box-and-whisker plot with median and range from minimum-to-maximum.To explore the effect of TNF-α on adhesion molecule and GRK mRNA expression in endothelial cells, we examine the online publicly available endothelial cell database (EndoDB) (endodb/) , which c5 cells per well, grown overnight and switched to low serum (0.4% FBS) for 24 h 37°C. Cells were washed, incubated with DMEM containing 10 mM HEPES, 1 mM CaCl2, and 1 mg/mL BSA for 1 h and then pretreated with 20 nM APC for 3 h followed by TNF-α (10 ng/mL) stimulation for 24 h at 37 °C. Cells were lysed in RIPA buffer  supplemented with protease inhibitors . Equivalent amounts of cell lysates were immunoblotted with either VCAM-1, ICAM-1, GRK2,3, GRK4,5,6 or GAPDH antibodies, developed by chemiluminescence and quantified by densitometry analysis using ImageJ software.Endothelial EA. hy926 cells were seeded in 12-well plates at 2.8 × 105 cells per well, cultured and serum-starved and pretreated with 20 nM APC for 3 h, followed by incubation with TNF-α (10 ng/mL) for 24 h 37°C. Cells were then washed with PBS and then left untreated (0 h) or treated with 10 μg/mL Actinomycin D for 2, 4, 5 and 6 h at 37°C. RNA was extracted and VCAM1 mRNA transcripts quantified by RT-qPCR using a TaqMan Gene Expression Probe  as described below.Endothelial EA. hy926 cells were seeded in 6-well plates at 3.2 × 105 cells per well in a 12-well plate, grown overnight, and transfected with siRNA using the TransIT-X2 System  according to the manufacturer’s instructions. Experiments were conducted 48 h post transfection. The following siRNAs were used in these studies: 25 nM GRK2 #1 siRNA 5′-CCGGGAGATCTTCGACTCATA-3′ (Qiagen #SI00287378), 25 nM GRK5 siRNA #5 5′-AGCGTCATAACTAGAACTGAA-3′ , 25 nM of PAR1-208 siRNA 5′-AGAUUAGUCUCCAUCAAUA-3′  according to according to the manufacturer’s instructions. RNA was quantified and cDNA synthesized from 1 μg RNA using SuperScript IV VILO Master Mix with ezDNase enzyme kit . Quantitative RT-PCR was performed with TaqMan Fast Advanced Master Mix  and TaqMan Gene Expression Probes including: PAR1 (F2R) , PAR3 (F2RL3) , VCAM-1 (VCAM1)  and TNF-α . The mRNA transcript levels were normalized to 18S expression and analyzed using the comparative CT (threshold cycle) method.Endothelial EA. hy926 cells were seeded in 6-well plates at 3.2 × 102 in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco #10–013-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco #10437–028). HEK293 cells were seeded at 2.5 × 105 cells per well of a 12-well plate, grown overnight, and then transfected with BRET donor RLuc-β-arr2 (100 ng) together with BRET acceptor PAR1-YFP (400 ng) and the APC co-receptor EPCR Halo (200 ng), with polyethylenimine (PEI) diluted in Opti-MEM. The next day, cells were pooled and re-seeded into poly-D-lysine coated 96-well plate at 3 × 104 cells per well and grown overnight. The following day, cells were washed with PBS and starved for 1 h using a 1:1 equal mixture of DMEM no phenol red  combined with PBS. After starvation, cells were preincubated with 5 µM of RLuc substrate Coelenterazine H  for 5 min followed by the addition of 20 nM APC  and BRET measurements were taken at 37°C over time. All BRET measurements were performed with a Berthold TriStar LB941 multimode plate reader running MicroWIN 2010 software (Berthold Biotechnologies) using two filter settings: 480 nm for Rluc and 530 nm for YFP. The BRET signal was calculated as the emission at 530 nm divided by the emission at 480 nm. The BRET signals were normalized to basal BRET ratios and expressed as the percent over basal.HEK293 cells were purchased from ATCC and maintained at 37°C in 5% COAPC-induced changes in phosphorylation of TNF-α signaling pathway components were determined from three biological replicates using tandem mass tag 10-plex quantitative phospho-proteomic analysis as we previously described . BrieflyBioRender.com. The “TNF Pathway” was adapted from BioRender.com (2023) https://app.biorender.com/biorender-templates.Adobe Illustrator and Photoshop were used to create figures. The cartoons were created with Data were analyzed using Prism software . Statistical analysis was determined by performing one-way analysis of variance (ANOVA) with multiple comparisons.A key feature of APC/PAR1 cytoprotective signaling is the capacity to attenuate TNF-α induced endothelial inflammatory responses . TNF-α, To understand how APC/PAR1 might modulate pathways associated with TNF-α inflammatory signaling, a quantitative phospho-proteomics mass spectroscopy analysis of APC stimulated human umbilical vein endothelial cell (HUVEC)-derived EA. hy926 cells was examined . EndotheCAM1, ICAM1 and SELE (E-Selectin) gene expression. We evaluated five independent datasets from studies using human cultured endothelial cells treated with TNF-α including four human umbilical vein endothelial cell (HUVEC) studies  and one aortic endothelial cell culture study . TNF-α induced VCAM1 gene expression in all five independent studies  and E-Selectin, that facilitate leukocyte recruitment and inflammatory responses. Induction of VCAM-1, ICAM-1 and E-Selectin by the cytokine TNF-α is mediated by AP-1, p38 MAPK and NF-κB pathways  Joyce e. Thus, w studies . A simil studies , support studies .VCAM1 mRNA stability. In these experiments, endothelial cells were pretreated with APC, stimulated with TNF-α and then incubated with actinomycin D to block gene transcription and the abundance of VCAM1 mRNA measured over time using RT-qPCR. In the absence of APC pretreatment, an approximately 50% loss of VCAM1 mRNA was detected after actinomycin D-mediated blockade of gene transcription  . Of the In summary, this study illustrates the complexity of APC/PAR1 β-arrestin-2 signaling that is controlled by different GPCR co-receptors and GRKs that drive distinct endothelial cytoprotective responses. Understanding APC/PAR1 signaling pathways that enable the endothelium to acquire resilience to resist injury and disruption is critical for the advancement of new targets for therapeutic development.


Utero-vaginal brachytherapy (BT) is an irreplaceable care component for the curative treatment of locally advanced cervix cancer (LACC). Magnetic Resonance Imaging (MRI)-image guided adaptive BT (IGABT) using the GYN-GEC-ESTRO EMBRACE guidelines is the international care standard. Usually following chemo–radiation therapy (CRT), IGABT has high proven utility in LACC but requires significant health system resources. Timely access was disrupted by the COVID-19 pandemic which challenged us to re-design our established IGABT care pathway.From April 2020 consecutive patients with LACC were enrolled after CRT in a single arm exploratory non-inferiority study of a modified IGABT (mIGABT) protocol. This delivered an iso-effective IGABT dose (39.3 Gy: EQD2: α/β10Gy concept) over a 24-h period during a single overnight hospitalisation.Fourteen LACC patients received mIGABT from April 2020 to March 2022. Median age was 62.5 years (37–82 years). LACC histology was primary squamous (9/14) or adeno-carcinoma (5/14). International Federation of Gynaecology and Obstetrics (FIGO) 2018 stages ranged from IB1/2 (N = 3), IIA1/IIB (5), IIIB (2), IIIC1/2 (4) with mean ± standard deviation (SD) gross tumour volume-at-diagnosis (GTV_D) of 37.7 cc ± 71.6 cc. All patients achieved complete metabolic, clinical, and cytologic cancer response with CRT and IGABT. High-risk HPV was cleared by 6-months. Complete MRI-defined cancer response before mIGABT (GTV_Fx1) was seen in 77% of cases (10/13). Only two women developed metastatic disease and one died at 12-months; 13 patients were alive without cancer at mean 20.3 ± 7.2 months follow-up. Actuarial 2-year overall survival was 93%. Compared with our pre-COVID IGABT program, overall mIGABT cost-saving in this cohort was USD 22,866. Prescribed dose covered at least 90% (D90) of the entire cervix and any residual cancer at time of BT  with 3-fractions of 8.5 Gy delivered over 24-h (22.8 ± 1.7 h). Total treatment time including CRT was 38 days. The mIGABT schedule was well tolerated and the entire cohort met EMBRACE recommended (EQD2: α/β10Gy) combined HRCTV_D90 coverage of 87.5 ± 3.7 Gy. Similarly, organ-at-risk (OAR) median: interquartile range D2cc constraints (EQD2: α/β3Gy) were EMBRACE compliant: bladder (65.9 Gy: 58.4–72.5 Gy), rectum (59.1 Gy: 55.7–61.8 Gy), and sigmoid colon (54.6 Gy: 50.3–58.9 Gy). ICRU recto-vaginal point dose was significantly higher (75.7 Gy) in our only case of severe (G4) pelvic toxicity.This study demonstrated the utility of mIGABT and VMAT CRT in a small cohort with LACC. Loco-regional control was achieved in all cases with minimal emergent toxicity. Single insertion mIGABT was logistically efficient, cost-saving, and patient-centric during the COVID-19 pandemic. External beam radiation therapy (EBRT) was delivered by dynamic single isocentre volumetric-modulated arc therapy (VMAT) on either Varian TrueBeam® or Halcyon® linear accelerators . Daily cone-beam computer tomography (CBCT) provided image guidance. The primary central pelvic planning target volume (PTV_P) included cervix and corpus uteri, parametria, and additional vaginal margin on any gross disease identified on staging MRI. Iodated hydroxyethyl cellulose vaginal contrast (Iopromide 5 cc and K–Y gel® 30 cc agitated in 60 cc catheter tip syringe) was routinely used to delineate the vagina from fornix to introitus during simulation  and reusable vacuum cushion   since 2010. Significant applicator displacement or pelvic shift during multiple patient transfers was minimal . ImmobilMRI/CT compatible applicators were of 2 types: Vienna Ring Set 26–34 mm (Elekta AB: Part No. 189.569) with fixed angle (60°) intrauterine 20-60 mm tandems (Tandem + Ring (T + R); 12/14 women: 86%), or Vaginal Applicator set (Elekta AB: Part No. 101001): 20-80 mm angled tandems (10–45°) and segmented (20-35 mm) cylinder set (Tandem + Cylinder (T + C); 2/14: 14%). No patient required a hybrid interstitial technique.A dedicated brachytherapy planning system  was used to contour, reconstruct, optimise, and evaluate dose target coverage. A standard loading pattern similar to a Manchester “Point A” plan was used as the starting point before manual conservative optimisation to ensure 3D-volume-based planning metrics were met. At least 90% of the ICRU 89 high-risk CTV (HRCTV) received 100% of the prescription dose (HRCTV_D90 concept) . Velocity® allowed rigid applicator registration and assessment of dosimetric uncertainties due to applicator displacement after Fraction 1. Oil-filled markers were placed within the applicators pre-imaging to aide localisation , and imported into Velocityl et al. , a toler192Ir . The EMBRACE/ICRU 89 IGABT protocol delivered a biologically-equivalent tumour dose (Linear-Quadratic [LQ] model: EQD2 α/β10Gy) of 39.7 Gy (4 IGABT fractions of 7 Gy in 2 insertions to the HRCTV_D90 over 8 days). We hypothesised a bio-effective mIGABT scheme to be 39.3 Gy (25.5 Gy in 3 fractions of 8.5 Gy in 1 day) as derived from the formula:Modified IGABT was delivered with HDR EQD2 mIGABT = Total modified dose (d x number fractions) x (α/β + d) (α/β + 2)−1 Gy, where d = modified dose (Gy)/fraction, and α/β for acute tumour effect = 10 Gy. EQD2 exposures for OAR late (injury) effects were derived at α/β = 3 Gy. coverage was 37.5 ± 3.5 Gy. Combined total mean ± SD and median (IQR) HRCTV_D90 coverage was 87.5 ± 3.7 Gy and 89.4 Gy (87.2–89.6), respectively.Complete MRI-defined cancer response before mIGABT (GTV_Fx1) was seen in 77% of cases (10/13). Measurable disease at fraction 2 was detected in 4 cases (GTV_Fx2): Patient 2 (21 cc), 4 (0.34 cc), 5 (4.4 cc), and 8 (1.3 cc). Minimal inter-fractional HRCTV adaption was required.® platform allowed rigid registration of pelvic simulator CT acquired in the brachytherapy facility and obviated the Fraction 3 MRI simulation and the associated in-patient bed transport to radiology.Dosimetric re-plans were necessary in only 3 patients . Contributors to this low frequency included geometric stability of patient-applicator setup, no or small residual GTV at time of mIGABT , and minimal perturbations in OAR volumes over 24 h. The VelocityAll patients achieved complete clinical, metabolic (FDG-PET), and cervical “co-test” response at 3 months. High-risk human papilloma virus (hr-HPV) was cleared in every case either at this point (11/14: 79%) or at 6 months follow-up . Although Patient 1 and Patient 5 progressed systemically, no significant local symptoms were recorded , or was detected on re-imaging after salvage chemotherapy (Patient 1). This implied durable LACC control within the pelvis. Two-year actuarial PFS was 86% (12/14) and overall survival 93% (13/14).Systemic metastatic disease occurred in 2 cases (Patient 1 and Patient 5). Both had unfavourable Stage IIIB SCC with significant vaginal disease. Time-to-metastatic event was 10 and 12 months respectively. Patient 5 developed peritoneal metastases and died of sepsis at 12 months post-mIGABT without further cancer-directed care. Patient 1 was diagnosed with a solitary (biopsy-proven) lung metastasis on routine re-staging FDG-PET at 12 months. Salvage cisplatin/paclitaxel doublet chemotherapy with bevacizumab  resultedIn this context, the cost of delivering IGABT (28 Gy/4 fractions) is 6,024.98 AUD and 3,756.49 AUD to deliver mIGABT (25.5 Gy/3 fractions). Adopting our mIGABT approach resulted in Public Health cost savings of 2,268.49 AUD  per treated patient (Table Both components of care (CRT + mIGABT) were well tolerated. Acute cysto-proctitis during CRT was usually minor (≤ G2) and self-limiting in 13/14 (93%). Excluding Patient 2 , median pelvis-only PTV V43Gy was 1360 cc (1322–1418.5 cc).Severe (≥ G3) acute toxicity was documented in Patient 2 only. As previously stated, this case had massive GTV_D (266.2 cc) and was also the only patient who required extended-field VMAT due to gross para-aortic lymph node metastases (Stage IIIC2). Final PTV V43Gy 2407.3 cc) and V55Gy (1117.5 cc) were both significant [.3 cc andOAR exposures at completion of treatment were well within ICRU 89 guidelines. Respective total median D2cc. EQD2α/β3Gy] for bladder, rectum, and sigmoid colon was 65.9 Gy (58.4–72.5), 59.1 Gy (55.7–61.8), and 54.6 Gy (50.3–58.9). Median dose at ICRU dose points for bladder 62.9 Gy (52.3–79.6) and recto-vaginal (RV) septum was 66.2 Gy (53.6–80.7). By comparison, absolute ICRU RV dose in Patient 1 and Patient 2 was 87.8 Gy and 75.7 Gy respectively, after T + C brachytherapy. Table Gy for blIn LACC the vagina is both an OAR and potential target volume. Vaginal ICRU points reflected brachytherapy dose gradients at the level of vaginal sources 5 mm deep to the surface of the vaginal ring (12/14 cases) or cylinder dome . The exposures are representative at the mid-circumference within the 4 quadrants of the vaginal fornix. The T + C set did not allow differential (anisotropic) dosing and were excluded. Respective IGABT median (IQR) right, left, anterior, and posterior vaginal point exposures  were: 6.2 Gy (5.6–6.9), 5.8 Gy (4.9–7.1), 4.1 Gy (3.4–4.9), and 5.0 Gy (3.6–6.4). Total absolute doses were 52.5 Gy, 52.1 Gy, 50.4 Gy, and 51.3 Gy, respectively.Table Over a 15-year period a landmark series of Gynaecological (GYN) GEC-ESTRO Working Group Recommendations (I–IV) described a coherent methodology of utero-vaginal MRI-IGABT prescription, recording, and reporting in LACC –14. ThesBased on these principles, and through prior IGABT clinical practice , 47, theEMBRACE-I established the combined (EBRT/IGABT) minimal HRCTV_D90 target dose of 85 Gy [EQD2: α/β10Gy] as a hard goal for controlling the central pelvis. A range of safe OAR D2cc exposures (EQD2: α/β3Gy) were also reported. These have since been refined as primary planning aims in the current EMBRACE-II study . SpecifiDespite these gains, optimal treatment of LACC is technically complex and health resource consumptive. In LMICs with the highest prevalence of cervix cancer, sophisticated oncologic care is simply unavailable or difficult to access , 4, 6. FAlthough major healthcare disparities are rare in Australia , the 202This non-inferiority study of 14 women with LACC demonstrated the real-world “fungibility” of the radio-biological backbone of EMBRACE-I. Our IGABT fraction sizes of 8.5 Gy via a single utero-vaginal insertion delivered a mean (SD) combined HRCTV_D90 of 87.5 ± 3.7 Gy (EQD2Gy) in 3 fractions accelerated over 24 h. This schedule with pre-emptive or sequential CRT was generally well tolerated. All indices of HRCTV_D90 coverage, OAR, and most individual organ system ICRU dose point exposures were within published efficacy and chronic safety recommendations. Overall treatment time was significantly less than 50 days (38 days). Actuarial overall metabolic (FDG-PET) progression-free survival was 93%. All patients achieved central pelvis control with early eradication of malignant cytology (3-months) and hr-HPV (by 6-months) on PCR co-testing. Lymph node failure or persistent nodal metastatic disease did not occur. Our EBRT induction platform of dynamic VMAT and simultaneous accelerated integrated boost (55-60 Gy) to GTV_N with full chemotherapy intensity in 93% of patients (13/14) is assumed to have contributed to this success . RegistrOur experience paralleled that of Mahantshetty et al. . Their PAlthough a post-COVID-19 change in our pattern of brachytherapy practice awaits longer follow-up, limitation to our method was suggested by significant late toxicity signals in one patient (Patient 2) with advanced central pelvic cancer (GTV_D: 266.2 cc). Extensive residual upper and middle third vaginal disease after CRT (HRCTV: 68 cc.) required a less optimal T + C IGABT technique which did not permit differential loading at the cervix level. The final combined ICRU RV point dose was almost 137% higher than the cohort median (75.7 Gy vs. 55.3 Gy) despite a low cancer residuum within the cervix (24 cc). ICRU Point A dosing was also highest  who is cancer-free at 30-months post-anterior exenteration, implicated both large residual HRCTV, history of cigarette smoking, and mIGABT applicator type to risk of organ-system injury.


However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for in vitro N-glycosylation reactions. For this purpose, transmembrane-deleted human β-1,2 N-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and β-1,4-galactosyltransferase  have been expressed in Escherichia coli and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the N-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from Trypanosoma brucei.Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant oligosaccharyltransferases can be applied to glycosylate “empty,” i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to   N-linked glycans occur in all domains of life and can affect protein stability, solubility, cell trafficking, signaling, antigenicity as well as immunogenicity    isoforms . These i jejuni) . PglB trin vitro . Howeverow (<1%) .in vitro synthesis of novel synthetic LLOs and the transfer thereof to synthetic peptides are shown. For the former we used a set of recombinant Endoplasmic reticulum-resident glycosyltransferases  and a set of Golgi-resident glycosyltransferases , α-1,6-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT2ΔTM), β-1,4-galactosyltransferase 1 , both expressed in E. coli or S. cerevisiae (N-glycosylation consensus sequence Asn-Xaa-Thr/Ser (Xaa ≠ Pro) to a peptide comprising 10 amino acids, by recombinant Trypanosoma brucei STT3A  according to Rexer et al. (2020) (The plasmid for ALG1ΔTM was transferred into and expressed by . (2020) .6-tag) for purification. Vectors were transformed in Shuffle® T7 Express lysY (MGAT2ΔTM) and BL21 (DE3)  E. coli cells, respectively. Positive transformants, selected by antibiotics, were used for protein expression. In general, cultures were cultivated in terrific broth medium under agitation at 30°C. Overnight cultures were inoculated to an optical density at 600 nm (OD600) of 0.1. Gene expression was induced at OD600 = 0.6 by the addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cultures were cooled to 16°C. Cells were harvested after overnight incubation by centrifugation  and cell pellets were stored at −20°C until further usage.Gene sequences of human MGAT1ΔTM and GalTΔTM were inserted into the expression vector pET-28a (+); the gene sequence of MGAT2ΔTM was inserted into the expression vector pET-28b (+). Both vectors harbor an N-terminal 6-fold histidine tag . Cells were harvested after 23 h of cultivation  by centrifugation , washed once with ice cold water and cell pellets were stored at −20°C. 3 g wet biomass were resuspended in 30 mL buffer A (30 mM Tris (pH 7.5), 3 mM MgCl2, 0.1% octylphenoxy poly (ethyleneoxy) ethanol (IGEPAL CA-630) and lysed at 1,000 bar for three cycles. Cell debris was removed by centrifugation for 20 min at 8,000 x g (4°C), followed by ultracentrifugation . Membrane fractions were solubilized in 13.5 mL buffer A with 50% (v/v) glycerol and used for all synthesis reactions. This is chromatographic purification of Alg2 did not yield pure, active enzyme solutions  using the yeast strain Yeast ORF ALG2 (YGL065C), which was purchased from the Dharmacon™ Yeast ORF Collection  . For genolutions .T. brucei coding for STT3A with a 10-fold histidine and yellow fluorescent protein tag was purchased from Thermo Fisher Scientific.STT3A expression and purification were performed according to g, 20 min, 4°C). Supernatants were then applied to an equilibrated immobilized metal affinity chromatography (IMAC) column . Purified enzyme solutions were desalted using Amicon® Ultra 0.5 mL filters from Merck  with a molecular weight cut-off of 10 kDa according to the manufacturer’s instruction. Desalted enzymes were stored in 50% (v/v) glycerol at −20°C.All enzymes with the exception of Alg2 were purified. For purification, cells were lysed by high-pressure homogenization  followed by centrifugation . Typically, 10 µL aliquots were taken from reaction batches for analysis by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) analysis. Glycan nomenclature is detailed in 2 (buffer B), 1 mM dithiothreitol, 0.1 mM phytanyl-PP-chitobiose, 2 mM GDP-mannose, 0.1 mg/mL purified ALG1ΔTM and 35% (v/v) ALG2 yeast membrane fraction. After 8 h, the reaction batch was quenched for 5 min at 90°C to ensure enzyme inactivity.This one-pot multi-enzyme run was carried out for 8 h. The reaction batch contained: 50 mM 3-(Morpholin-4-yl)propane-1-sulfonic acid (pH 6.8), 0.1% IGEPAL CA-630, 10 mM MgCl2 (buffer C) with 4 mM UDP-N-acetylglucosamine (UDP-GlcNAc), 50% (v/v) LL-Man3 and 1 mg/mL MGAT1ΔTM. After 24 h, reactions were quenched for 5 min at 90°C. The second reaction, to synthesize LL-Man3GlcNAcGal1[3] was conducted in buffer C, 4 mM UDP-galactose, 50% (v/v) LL-Man3GlcNAc1[3] and 0.84 mg/mL GalTΔTM. The reaction was performed for 24 h and was quenched at 90°C for 5 min.The first reaction contained 25 mM HEPES (pH 7), 0.1% IGEPAL and 10 mM MnClSequential synthesis: The first reaction batch contained buffer C with 4 mM UDP-GlcNAc, 50% (v/v) Man3 and 1 mg/mL MGAT1ΔTM and MGAT2ΔTM, respectively. After 24 h, reactions were quenched at 90°C for 5 min and centrifuged. The second reaction batch contained: buffer C, 4 mM UDP-galactose, 0.84 mg/mL GalTΔTM and 50% (v/v) LL-Man3GlcNac2. The reaction was quenched at 90°C for 5 min after 24 h of incubation.One-pot synthesis: The one-pot synthesis of LL-Man3GlcNac2Gal2 was performed in buffer C with 4 mM UDP-GlcNAc, 4 mM UDP-Gal, 35% (v/v) LL-Man3 and 1 mg/mL MGAT1ΔTM, MGAT2ΔTM and GalTΔTM, respectively. The reaction was performed for 36 h at 30°C under agitation (350 rpm). After 0, 4, 8, 24 and 36 h, aliquots of 10 µL were taken and the reaction was quenched for 5 min at 90°C.N-Y-T-Y-T-Q, were purchased from Biomatik . The reaction was conducted in a volume of 100 µL and contained: 20 mM HEPES (pH 7.5), 10 mM MnCl2, 150 mM NaCl, 0.035% (w/v) DDM, 0.007% (w/v) CHS, 50% (v/v) LLO, 20 µM synthetic peptides, 1.8 mg/mL T brucei STT3A and EDTA-free protease inhibitor .To analyze the transfer of LLOs 5-carboxytetramethylrhodamine (TAMRA)-labeled synthetic peptides with the consensus sequence Asn–Xaa–Thr/Ser and the following amino acid sequence, G-S-D-A-The synthesis of LLOs was analyzed by xCGE-LIF . Glycans® Ultra 0.5 mL filters; Merck, Darmstadt, Germany). Samples were desalted by manual C18 solid-phase extraction. Therefore, samples were reconstituted in up to 1 mL 0.1% trifluoroacetic acid (TFA) to ensure that pH is lower than 3. HyperSEP C18 columns (ThermoFisher Scientific) were conditioned with 3 mL of conditioning buffer . Prior to sample loading the column was equilibrated with 0.1% TFA in water. Samples were desalted using 0.1% TFA in water and glycopeptides were eluted with 50% acetonitrile in water containing 0.1% TFA. For detergent removal HiPPR™ spin columns (ThermoFisher Scientific) were used according to the manufacturer’s instruction. Sample measurement and glycoproteomic analysis were conducted as described previously (® software (Protein Metrics).Prior to the analysis by mass spectrometry, enzymes and larger molecules were separated from the reaction products by filtration using a 10 kDa molecular weight cut-off filters  was subjected to GalTΔTM and UDP-galactose. As depicted in One-pot synthesis of LL-Man3GlcNAc2Gal2: To reduce the complexity of the synthesis, the production of LL-Man3GlcNAc2Gal2 was also tested in a one-pot reaction. For this purpose all enzymes  were added at the start of the reaction. Samples were analyzed by xCGE-LIF after 4 h, 8 h, 24 h and 36 h reaction time. Results of the reaction are depicted in in vitro glycosylation runs were conducted, to investigate the ability of recombinant T. brucei STT3A to transfer the synthesized hybrid  and complex-type structures , from the phytanyl anchor to the asparagine residue of the N-glycosylation consensus motif of the selected peptides  of identified glycopeptide spectra after the in vitro glycosylation reaction are depicted in N-glycan structures were identified. The fraction of glycopeptides containing LL-Man3GlcNAc1[3] was around 30%  enzymatic methods are especially useful as they do not require complex modifications of host cells or time consuming bioreactor cultivations but can be applied independently using a range of bacterial and mammalian Leloir glycosyltransferases that are already available  of peptides. As substrates for the glycosylation reaction, lipid-linked glycans are required and a range of saturated and unsaturated synthetic lipid-linked carriers have been synthesized and successfully used to replace natural lipid carriers that are not soluble in water. Thus, the complexity of the in vitro reaction can be reduced significantly (in vivo eukaryotic OSTs transfer (high-)mannose-type glycans, their use for transfer of hybrid- and complex-type N-glycans in vitro has not been investigated. Here, for the first time, complex N-glycan moieties were synthesized on synthetic lipid carriers. In particular, four novel LLOs–LL-Man3GlcNAc1[3] LL-Man3GlcNAc1Gal1[3], LL-Man3GlcNAc2 and LL-Man3GlcNAc2Gal2—were synthesized. Analysis of the reaction batches demonstrated that all final products were present as the main component in the reaction mix. In the future, these new LLOs and derivatives, for example, sialylated structures, can be used to investigate a range of novel OSTs that are either obtained through protein engineering or enzyme mining for their potential to transfer hybrid and complex-type N-glycan structures (Escherichia coli, the availability and costs of activated sugars and lipid-linked chitobiose are additional barriers for the synthesis of these LLOs at preparative scale. In particular, lipid-linked chitobiose is not commercially available and can only be obtained through elaborate chemical synthesis (To investigate key properties of therapeutic peptides and proteins, methods to generate homogeneous and defined glycoforms efficiently would be very helpful . In vitrvailable . In addivailable . Howevern system . Hence, ficantly . Howeverructures . Howeverynthesis .In vitro N-glycosylation reactions using recombinant T. brucei SST3A demonstrated that the structures Man3GlcNAc1[3] and Man3GlcNAc1Gal1[3] as well as the complex structure GlcNAc2Man3GlcNAc2 can be transferred to synthetic peptides. In comparison to other studies, however, the fraction of glycosylated over aglycosylated peptides after the reaction was very low (in vitro glycosylation reactions. Additionally, purified human Alg2 expressed in HEK cells lines can be used as alternative to avoid LL-Man5 formation by residual Alg11 (in vitro glycosylate longer and more complex peptides, or even proteins, should be determined in future studies.very low . As prevvery low . As expeal Alg11 . Furtherin vitro glycosylation of aglycosylated peptides at the eukaryotic N-glycosylation consensus sequence with hybrid and complex structures using chemoenzymatically preassembled LLOs.Altogether, the presented results offer a new synthetic approach for the


This complex is very stable toward dioxygen due to steric protection of the nickel(II) center. Here, we report the dioxygen reactivity of a new Ni–NO complex, [Ni(NO)(I)(L1″)], with a less hindered N2 type bis(pyrazolyl)methane ligand, which creates a coordinatively unsaturated ligand environment about the nickel center. Here, L1″ denotes bismethane. This complex is also described as a hs-nickel(II) center with a bound 3NO−, based on spectroscopic and theoretical studies. Unexpectedly, the reaction of [Ni(NO)(I)(L1″)] with O2 yielded 2-O2N)(L1″)2](I3) (L3 SNO = 1/2) to 1NO+ , and to NO−  when binding to a transition metal ion, i.e., Mn+–NO·, Mn−1–NO+, and Mn+1–NO−  complex.Nitrogen monoxide (·NO) is a small, relatively unstable, potentially toxic, diatomic free-radical molecule. ·NO has become one of the most studied and fascinating molecules in biological as well as inorganic chemistry ,7,8,9,10Mn+1–NO− ,13. By cMn+1–NO− . In the 6F5)(L1)]  ,16. This1)] − in ), and it1)] − in ,16. We h1)] − in . Later, omplexes ,10 with omplexes , Co 21]6F5)(L1)]omplexes , and Cu omplexes . Using dectively ,21,22. T· ligand ,10,23.10 system, did not show any reactivity toward dioxygen  and [Cu(κ1-ONO)2(L1″)]. The main differences between these complexes relate to the different ligand charges  and bulkiness (tertiary butyl versus isopropyl substituents)  and [CuCl2(L1″)], which again differ for the same reasons mentioned above  (PF6)       , an hs-{NiNO}dioxygen . This isdioxygen   . We alsoed above ,26. Thised above , [Ni(NO)ed above  (L0f− = NO)(L0)] ,29 (L0− n)](PF6)  (iPr3tacmp-tol)]  (Tmp-tolTseMes)]  (TseMes−BuIm)3}]  (HB(tBuIH2Im)3}]  (PhB(CyCMeBz)3}]  (HB(MeBzPhTz)3}]  (HB(p-tB3 group [[Ni(NO)(I)(L1″)] with a less hindered N2 type bis(pyrazolyl)methane coligand to create a coordinatively unsaturated ligand environment. We obtained an unexpected, six-coordinate compound, 2-O2N)(L1″)2](I3)[Ni(L3)], [Ni(NO)(L0f)] is also stable toward dioxygen due to the hindered CF3 group . In this3)(PPh3)2] was reported in 1971 by Professor Enemark , was obtained using [Ni(NO)(Br)(PPh3)2] as the precursor [3)2] with the potassium salt of the ligand [n could also be used for the synthesis [2(I)]2 and [Co(NO)2(I)]n were used to synthesize M–NO complexes such as [Fe(NO)(L3)] [2(L1″)](BF4), and [Co(NO)2(L1″)](BF4) [3)2] and [Ni(NO)(I)]n [n as starting material in this research. The Ni–NO complex [Ni(NO)(I)(L1″)] was obtained by the reaction of [Ni(NO)(I)]n and the ligand L1″, as shown in [Ni(NO)(I)(L1″)], suitable for structural characterization. The coordinated iodide ion is needed for the structural stability of the complex. If we add a THF solution of AgPF6 to a solution of [Ni(NO)(I)(L1″)] to remove the iodide ion, the color of the solution changes from green to colorless, indicating that, upon removing the iodide, the complex decomposes.The first structural characterization of a Ni–NO complex [Ni(NO)(L3)] , [Fe(NO)″)](BF4)  previousNO)(I)]n . Therefo[Ni(NO)(I)(L1″)] is shown in The structure of [Ni(NO)(I)(L1″)] is four-coordinate with three nitrogen atoms from L1″ and NO and the iodide bound. The Ni1–N11 and Ni1–N21 bond lengths (2.0157 (12) and 2.0234 (12) Å) are experimentally equivalent and considerably longer than the Ni1–N1 (1.6467(16) Å) bond. The N1–O1 bond length is 1.136 (3) Å and the Ni1–N1–O1 angle is 172.42(17)°. The presence of the bidentate ligand, with a restricted bite angle, introduces significant distortions in this coordination geometry. Thus, the variation in the N–Ni1–N angles is substantial, with the narrowest angle being 91.41 (5)° for N11–Ni1–N21 and the widest being 123.86 (6)° for N1–Ni–N11. The value of τ4 = [360 – (α + β)/141] = 0.80, where α and β are the two widest angles subtended at the metal center [τ4 in [Ni(NO)(I)(L1″)] is closest to 0.85, which corresponds most closely to a trigonal pyramidal geometry (C3v). In this structure, the three nitrogen donors form the basal  plane. To investigate this further, we calculated the distances between the basal planes formed by three of the coordinating atoms and the nickel center. Here, the distance between Ni1 and the plane formed by N11, N21, and I1 is much longer (1.0817 (3) Å) compared to the other three: 0.4900 (3) Å between Ni1 and the plane formed by N11, N21, and N1; 0.5673 (3) Å between Ni1 and the plane formed by N11, N1, and I1; and 0.5493 (3) Å between Ni1 and the plane formed by N21, N1, and I1. The latter three distances are quite similar. Based on these considerations, the coordination geometry of the complex is distorted tetrahedral, which is a different conclusion than the result from the τ4 value discussion.The coordination geometry of Ni in l center . The val−1 in these compounds, showing a larger degree of variability and indicating distinct differences in the electronic structures of these complexes. Therefore, careful spectroscopic experiments as well as theoretical support are very important to describe the true electronic structure of [Ni(NO)(I)(L1″)], as well as the oxidation state of the metal center and the NO group  were measured using KBr and CsI pellets, respectively (I)]n (−1 in [Ni(NO)(L0f)] [−1 in [Ni(NO)(L3)] [−1 in [Ni(NO)(I)(L1″)], as listed in −1, and is assigned considering related values, 574 cm−1 in [Ni(NO)(L3)] [−1 in [Ni(NO)(L0f)]  [IR and far-IR spectra of ectively . The ν(I)]n . ConsideO)(L0f)]  > 1780 cNO)(L3)]  > 1777 cNO)(L3)]  and 598 O)(L0f)]  (L0f)] .[Ni(NO)(I)(L1″)] are shown in d–d transitions [6F5)(L1)], the band at 804 nm is assigned to the 3T1(F) → 3T1(P) transition, giving rise to a pseudo A signal in MCD (magnetic circular dichroism) because of instate spin–orbit coupling [[Ni(NO)(I)(L1″)] exhibits strong absorption bands in the 500–700 nm range, as shown in d–d transitions of the high-spin Ni(II) center. Therefore, the UV–Vis absorption data indicate that the Ni oxidation number is +II, and that the Ni(II) center is in the high-spin state. This implies that the NO ligand is reduced in the complex to 3NO− (nitroxyl), since the complex charge is zero. This is further discussed in the DFT section. The complex [Ni(NO)(I)(L1″)] has a green color {λmax/nm (ε/M−1cm−1): 492 (130) and 717 (400)}, whereas [Ni(NO)(L3)] {λmax/nm (ε/M−1cm−1): 630 (480)} [λmax/nm (ε/M−1cm−1): 593 (440)} [[Ni(NO)(I)(L1″)] versus [Ni(NO)(L3)]), causing a weaker overall ligand field and a shift of the 3T1(F) → 3T1(P) type d–d transition to lower energy in [Ni(NO)(I)(L1″)] (717 nm) compared to [Ni(NO)(L3)] (630 nm) and [Ni(NO)(L0f)] (593 nm). These spectral features are different from those of the starting material [Ni(NO)(I)]n (UV–Vis spectra (dichloromethane solution and solid state) of nsitions . In [Ni}  and [Ni(3 (440)}  are blueNO)(I)]n .1H and 13C) are compiled in [Ni(NO)(I)(L1″)] has relative sharp 1H-NMR signals in both CDCl3 and (CD3)2CO solution, as shown in [Ni(NO)(I)(L1″)] is +II, and the metal center is in the high-spin state. Therefore, the total spin of the Ni(II) center should be S = 1. However, the NMR spectra clearly indicate that the complex is diamagnetic . This originates from antiferromagnetic coupling between the d-electrons of Ni(II) and the π*-electrons of the nitroxyl ligand . This assignment was previously proposed for other complexes, listed in Nuclear magnetic resonance (NMR) spectra ([Ni(NO)(I)(L1″)], on the other hand, reacts with dioxygen at room temperature ([Ni(NO)(I)(L1″)] and dioxygen are shown in [Ni(NO)(I)(L1″)] slowly reacts with dioxygen. We isolated the product and grew crystals. Crystallographic data of the product show that an unexpected six-coordinate structure was obtained, with the oxidation of 3NO− and I− to NO2− and I3−, as shown in [Ni(NO)(I)(L1″)] decomposed, and L1″ itself was reconstituted. The product complex has a six-coordinate structure with two L1″ ligands bound to the nickel ion and one coordinated NO2−, which originates from the oxidation of 3NO−. The complex is paramagnetic, as evident from 1H-NMR spectroscopy, and therefore contains a hs-Ni(II) center.As mentioned above, the coordinately saturated Ni–NO complexes [Ni(NO)(L3)] and [Ni(NO)(L0f)] do not react with dioxygen. The coordinately unsaturated Ni–NO complex perature . During 2-O2N)(L1″)2](I3)(I3)(I3)[Ni stretches of the coordinated NO2− group (s(N–O) stretch of the NO2− group in [Cu(κ2-ONO)(L3)] was observed at 1264 cm−1 (I3)[Ni] ]  (I3)[Ni(L1″)2](I3) complex using def2-TZVP as the basis set and different functionals. The results are shown in 10, but for other {MNO}x systems as well , we therefore investigated whether the analogous open-shell singlet electronic state could be geometry optimized. Excitingly, using B3LYP we were able to obtain a fully optimized structure of the complex in the BS state. The resulting wavefunction shows spin densities of +1.05 on Ni(II) and −1.19 on the NO− ligand. A superposition of the calculated BS structure and the crystal structure, shown in In these calculations, a closed-shell (CS) electron configuration was used. However, experimentally, the related complex [Ni(NO)(L3)] has been shown to have an open-shell ground state, with a hs-Ni(II) center  . Neverth[Ni(NO)(I)(L1″)] in the BS state is about 2 kcal/mol lower compared to the CS system. Given the similarities between the structural and spectroscopic properties of [Ni(NO)(I)(L1″)] and [Ni(NO)(L3)] and the detailed investigations on the latter complex [[Ni(NO)(I)(L1″)], as observed for [Ni(NO)(L3)]. Attempts to geometry-optimize the complex in the BS state using BP86 were not successful, as the wavefunction always collapsed into the CS state. Comparing the results obtained for the open-shell (BS) and CS systems using the B3LYP functional, we see that for the BS state, the Ni–NO bond distance is longer and ν(Ni–NO) is lower compared to experiment. In contrast, for the CS state, the Ni–NO distance is underestimated and ν(Ni–NO) is overestimated. These results show that the experimental Ni–NO bond strength is in between the predictions for the open-shell and closed-shell system. On the other hand, the Ni–N–O bond angle and the N–O stretching frequency obtained for the BS state are closer to the experimental values. The calculated energy for  complex , these r2-O2N)(L1″)2]+[Ni(I)(L1″)] with O2. In this case, the complex does not involve an antiferromagnetically coupled ground state. For this reason, optimization and frequency calculations were performed using the functional BP86, since it shows the overall best results for the Ni–NO complex. The results from these calculations are shown in In order to assist with the experimental findings, DFT calculations were also performed for the −1, which shift to 1247/1241 cm−1 upon 18O isotope labeling . On the other hand, the antisymmetric N–O stretch of coordinated nitrite is predicted at 1167 cm−1, shifting to 1147 cm−1 with 18O. The experimental value for the N–O stretch is observed at 1201 cm−1, which is right in between those predicted frequencies. However, the experimentally observed isotope shift of 19 cm−1 , and NO gas was purified by passing through a column filled with solid NaOH before the reaction. 18O2 gas was obtained from Shoko Science and was used without any purification . Other reagents were commercially available and used without further purification. The bismethane (L1″) was prepared following a published method  was obtained from crystallographic data by removing the tetrahydrofuran molecule present in the structure. The initial structure for 2-O2N)(L1″)2][Ni [[Ni(NO)(I)(L1″)], we performed both closed-shell and broken-symmetry calculations.Computational studies were performed using Gaussian 16 software . The geof2-TZVP) ,49. The f2-TZVP)  and the f2-TZVP) ; (ii) Bef2-TZVP) , along wf2-TZVP) . For 19H32IN5NiO·0.5(H2O): C 42.89, H 6.06, N 13.16. Found: C 41.94, H 5.87, N 12.77. IR (KBr/cm−1): 2965 s, 2933 m, 2869 s, 1776 vs ν(N–O), 1547 m, 1461 m, 1437 m, 1402 w, 1382 m, 1366 w, 1281 s, 1181 m, 1053 m, 809 m, 796 m. Far-IR : 661 s, 571 m, 543 w, 470 w, 410 w δ(Ni–N–O), 381 m, 329 w, 303 w, 252 w, 212 w, 188 w, 163 w. 1H NMR (CDCl3) δ/ppm (assignment): 1.25 2), 1.40 2), 1.67 2), 2.75 2), 3.83 2), 4.02 , 6.24 . 1H NMR (acetone-d6) δ/ppm (assignment): 1.30 3), 1.47 3), 1.71 3), 3.23 3), 3.85 3), 3.95 , 6.60 . 13C NMR (CDCl3) δ/ppm (assignment): 22.9 (CH(CH3)2), 25.7 (CH(CH3)2), 29.2 (CH(CH3)2), 53.1 (H2C), 100.6 (pz-4C), 152.4 (pz-3C), 164.7 (pz-5C). UV–Vis  λmax/nm (ε/M−1cm−1): 290 (4060), 375 (340), 492 (130), 717 (400). UV–Vis  λmax/nm: 297, 383, 500, 646, 730.Anal. Calcd for C[Ni(NO)(I)(L1″)]  was dissolved in dichloromethane (9 mL) at room temperature in an argon atmosphere. The argon was then replaced with O2 gas and the solution was stirred at room temperature for 3 days. During this time, the color of the solution changed from deep green to brown. Recrystallization from dichloromethane/n-heptane at −30 °C gave brown crystals. Single crystals suitable for X-ray diffraction were obtained by slow recrystallization under the same experimental conditions. Yield: 45% . In a 50 mL Schlenk tube, 38H64I3N9NiO2: C 40.81, H 5.77, N 11.27. Found: C 40.67, H 5.77, N 11.27. IR (KBr)/cm−1(assignment): 3123 w, 2967 vs, 2930 s, 2870 m, 1549 s, 1475 s, 1459 s, 1442 s, 1383 s, 1366 m, 1336 w, 1279 vs, 1201 s(ν(NO2)), 1183 s, 1055 m, 1016 w, 812 w, 661 w. Far-IR (CsI) cm−1: 672s, 662 s, 584 w, 539 m, 508 w, 475 w, 413 w, 391 w, 320 w, 311 w, 228 w, 182 w, 138 vs. UV–Vis  λmax/nm (ε/M−1cm−1): 295 , 365 . UV–Vis  λmax/nm: 301, 376.Anal. Calcd for C18O2 reaction, [Ni(NO)(I)(L1″)]  was dissolved in dichloromethane (5 mL) at room temperature in an argon atmosphere. The argon was then replaced with 18O2 gas and the solution was stirred at room temperature for 3 days. After the reaction, the recrystallization was carried out using the above procedure.For the −1(assignment): 3125 w, 2968 vs, 2931 s, 2870 m, 1550 m, 1473 m, 1459 m, 1441 m, 1385 s, 1366 m, 1335 w, 1280 vs (ν(N18O16O)), 1182 m, 1095 w, 1079 m, 1055 m, 1018 m, 810 m, 661 w.IR (KBr)/cm[Ni(NO)(I)(L1″)] and 2-O2N)(L1″)2](I3) with the less hindered N2 type bis(pyrazolyl)methane coligand L1″, to create a coordinatively unsaturated ligand environment, where L1″ denotes bismethane. The coordination geometry in [Ni(NO)(I)(L1″)] is four-coordinate with three nitrogen atoms from L1″ and NO and a bound iodide anion. We could conclude that the coordination geometry about the Ni center is distorted tetrahedral, based on distances between the basal planes and the metal ion. The ν(N–O) value is 1777 cm−1 in [Ni(NO)(I)(L1″)], which is clearly shifted from ν(N–O) in the starting material [Ni(NO)(I)]n, observed at 1854 cm−1. Moreover, the Ni–NO stretching vibration, ν(Ni–NO), is observed at 572 cm−1. This assignment is based on the consideration of the vibrational frequency of ν(Ni–NO) in related complexes, especially [Ni(NO)(L3)]. Considering the electron-donating properties of hydrotris(pyrazolyl)borate type ligands, the overall electronic structure of the related complexes [Ni(NO)(L3)] and [Ni(NO)(I)(L1″)] is almost the same, in spite of the different coordinating groups (pyrazolyl nitrogen versus iodide anion) and different ligand charges . From the UV–Vis assignments, the Ni oxidation state is +II, and the Ni(II) center is in the high-spin state. This implies that the NO ligand is 3NO− (nitroxyl) in the complex, since the complex charge is zero, which is analogous to the electronic structure reported for [Ni(NO)(L3)]. In comparison, [Ni(NO)(I)(L1″)] has a green color, not blue like [Ni(NO)(L3)]. This difference relates to the different ligand donor set (N3I1 versus N4), causing a shift in the low-energy d–d transitions in [Ni(NO)(I)(L1″)].Previously, we reported a Ni–NO complex with a hindered N3 type coligand, [Ni(NO)(L3)], where L3l)borate . This co[Ni(NO)(I)(L1″)], we obtained an unexpected six-coordinate compound, 2-O2N)(L1″)2](I3)[Ni(I)(L1″)] to O2, which can be attributed to one of the ν(N–O) modes of the bound NO2− group. For the anion I3−, the νas(I–I) band was observed at 137 cm−1, and characteristic strong bands in the UV region of the absorption spectrum were observed at 295 nm  and 365 nm , which are assigned as σ → σ* and π → σ* transitions of I3−, respectively.After dioxygen reaction of [Ni(NO)(I)(L1″)] using both closed-shell and broken-symmetry electronic states were conducted using different DFT methods. Notably, using B3LYP, we were able to optimize the structure of the complex for both of these electronic states, allowing us to make direct comparisons. The results suggest that the complex is best described as a hs-Ni(II) center (S = 1) antiferromagnetically coupled to the 3NO− ligand , with the broken-symmetry state being about 2 kcal/mol lower in energy than the closed-shell state. The electronic structure of [Ni(NO)(I)(L1″)] is therefore analogous to that of [Ni(NO)(L3)] reported previously, in agreement with their very similar structural and spectroscopic properties. DFT calculations for the six-coordinate product complex 2-O2N)(L1″)2]+[Ni(κ were also performed. Our results suggest that the N–O stretching mode of nitrite, observed experimentally at 1201 cm−1, corresponds to the antisymmetric stretch, predicted at 1167 cm−1 with a calculated 18O isotope shift of about 20 cm−1, in agreement with experiment.Theoretical calculations for


N-cyclohexyltryptamine is presented, along with those of its bromide and fumarate salts.The crystal structure of the freebase of the monoalkyl tryptamine  N-cyclohexyltryptamine (I) {systematic name: N-[2-(1H-indol-3-yl)ethyl]cyclohexanamine}, C16H22N2, and two of its salts, N-cyclohexyltryptammonium bromide (II) {systematic name: N-[2-(1H-indol-3-yl)ethyl]cyclohexanaminium bromide}, C16H23N2+·Br−, and N-cyclohexyltryptammonium fumarate (III) (systematic name: bis{N-[2-(1H-indol-3-yl)ethyl]cyclohexanaminium} (2E)-but-2-enedioate), 2C16H23N2+·C4H2O42−, were determined by single-crystal X-ray diffraction. The freebase compound forms infinite chains along [010] through N—H⋯N hydrogen bonds. The bromide salt is held together by N—H⋯Br interactions in two-dimensional sheets along (001). The fumarate salt is held together in infinite three-dimensional frameworks by N—H⋯O hydrogen bonds.The solid-state structures of  Tryptamine and its derivatives are an important class of biologically active compounds that are found in almost all organisms on Earth. In humans these compounds play significant roles ranging from the function of the gastrointestinal tract to neurotransmission and control subjective phenomena like happiness. The most abundant of these compounds, occurring naturally in the body, are primary tryptamines like tryptamine itself and serotonin  , 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 4-hydroxy-N,N-dimethyltryptamine (psilocin) which are known agonists of the serotonin 2A (5-HT2A) receptor and elicit a psychedelic response in humans. These and similar compounds have attracted a great deal of interest due to their potential for treating conditions including depression  receptor  tryptamines, including the natural products i.e. the monoalkyltryptamines; many of these compounds have been observed as natural products in plants. One study suggests that monoalkyltryptamines are generally less toxic than their dialkyltryptamine counterparts  and N,N-dimethyltryptamine (DMT) in mice were 78 and 43 mg kg−1, respectively. Recent studies have suggested that the psychedelic effects of compounds may not be necessary for the expression of therapeutic effects  are agonists of 5-HT2A but do not produce head-twitch response (HTR) in mice, which is characteristic of classic psychedelics such as psilocybin and LSD  and selectivity five times more potent (EC50 = 0.22 nM) than the migraine drug naratriptan (EC50 = 1.6 nM)  is shown in Fig. 1II), top right], and its fumarate salt . The asymmetric unit of (I) contains one full tryptamine (C16H22N2) molecule. The asymmetric unit of the bromide salt (II) contains one N-cyclohexyltryptammonium (C16H23N2+) cation and one bromide anion held together with an N2—H2A⋯Br1 hydrogen bond. The asymmetric unit of the fumarate salt (III) contains one full N-cyclohexyltryptammonium (C16H23N2+) cation and one half of a fumarate (C4H2O42–) dianion, with the second half generated by inversion. The two ions are connected in the asymmetric unit through a N2—H2⋯O2 hydrogen bond. The fumarate dianion is near planar, with an r.m.s. deviation from planarity of 0.011 Å. In all three structures, the cyclohexyl group is in a chair configuration. Table 1The molecular structure of the freebase of 3.N-cyclohexyltryptamine is shown in Fig. 2In the freebase, the tryptamine molecules are held together in infinite chains along [010] by N1—H1⋯N2 hydrogen bonds Table 2. In the 4.et al., 2022et al., 2020bN-methyltryptamine as a chloride salt  suitable for X-ray diffraction studies were grown by slow evaporation of an ethanol solution of a commercial sample (ChemBridge).Crystals of N-cyclohexyltryptamine (I) by stirring it in a biphasic mixture of dichloroethane and aqueous sodium hydroxide. The organic layer was isolated, washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to yield the freebase as a white powder. Crystals suitable for X-ray diffraction were grown by the slow evaporation of an acetone solution.The bromide salt was converted to freebase N-cyclohexyltryptamine and fumaric acid were dissolved in methanol and heated at reflux for 12 h. The solvent was removed in vacuo to yield an off-white powder which was characterized by NMR. Single crystals of (III) suitable for X-ray diffraction studies were grown from the slow evaporation of a methanol/water solution. 1H NMR : δ 7.55 , 7.35 , 7.21 , 7.07 , 6.99 , 6.43 , 3.08 , 2.99 , 2.89 , 1.98 , 1.72 , 1.19 .Freebase 6.A and H2B in the bromide salt, and H1, H2A and H2B in the fumarate salt were found from difference-Fourier maps. These hydrogen atoms were refined isotropically, using DFIX restraints with N—H(indole) distances of 0.87 (1) Å and N—H(amine/ammonium) distances of 0.90 (1) Å. Isotropic displacement parameters were set to 1.2 Ueq of the parent nitrogen atoms. All other hydrogen atoms were placed in calculated positions.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989023006217/tx2070sup1.cifCrystal structure: contains datablock(s) I, II, III. DOI: 10.1107/S2056989023006217/tx2070Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023006217/tx2070IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989023006217/tx2070IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 2281772, 2281771, 2281770CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: