Hydroxyl modified PBO (HPBO) fiber shows better photostability and interfacial shear strength. 2-Hydroxyterephthalic acid (2-HTA), the monomer for the HPBO fiber, is usually synthesized by chemical method, which has poor space selectivity and high energy consumption. The enzymatic Kolbe–Schmitt reaction, which carboxylates phenolic substrates to generate hydroxybenzoic acids with bicarbonate/COpara-position of the benzene carboxyl group, known as enzymatic Kolbe–Schmitt reaction. 2,3-Dihydroxybenzoic acid decarboxylase from Aspergillus oryzae  were expressed in recombinant E. coli and showed highest activity. The yield of 2-HTA was 108.97 ± 2.21 μg/L/mg protein in the whole-cell catalysis. In addition, two amino acid substitutions, F27G and T62A, proved to be of great help in improving 2,3-DHBD activity. The double site mutation F27G/T62A increased the production of 2-HTA in the whole-cell catalysis by 24.7-fold, reaching 2.69 ± 0.029 mg/L/mg protein. Moreover, de novo biosynthetic pathway of 2-HTA was constructed by co-expression of 2,3-DHBD_Ao and 3-hydroxybenzoate synthase Hyg5 in S. cerevisiae S288C with Ura3, Aro7 and Trp3 knockout. The engineered strain synthesized 45.40 ± 0.28 μg/L 2-HTA at 36 h in the CO2 environment.The biosynthesis of 2-HTA was achieved by the innovative application of hydroxybenzoic acid (de)carboxylases to carboxylation of 3-hydroxybenzoic acid (3-HBA) at the 2 fixation.De novo synthesis of 2-HTA has been achieved, using glucose as a raw material to generate shikimic acid, chorismic acid, and 3-HBA, and finally 2-HTA. We demonstrate the strong potential of hydroxybenzoate (de)carboxylase to produce terephthalic acid and its derivatives with COThe online version contains supplementary material available at 10.1186/s13068-023-02413-0. Using CO2 as a C1 carbon source to synthesize value-added chemicals and materials is an attractive strategy to reduce atmospheric CO2. While CO2 is chemical stability and high energy is required to activate this substrate.CO2 fixation process, which carries out carboxylation of phenol at the ortho- and para-position at 125 °C, 100 atm [2, 30 ℃) and employs nonoxidative decarboxylase which is originally discovered to catalyze the decarboxylation of benzoic acid derivatives. These hydroxybenzoic acid (de)carboxylases have exquisite regioselectivity in ortho-position of the phenolic -OH group and have a remarkably broad substrate spectrum that phenol derivatives containing alkyl, alkoxy, halo and amino groups are tolerated [2 gas (∼ 30–40 bar) is used instead of bicarbonate and the carboxylation process is greatly promoted, as the carboxylation conversion of resorcinol is up to 68% [The Kolbe–Schmitt reaction is a famous COolerated . In addi2 fixation pathways are well-researched and the CO2 fixation enzymes are applied to construct the new biosynthetic pathways [Escherichia coli (E. coli), which produces succinate from acetyl-CoA with the fixation of two CO2 molecules [2 Calvin–Benson–Bassham cycle that converts the yeast Pichia pastoris into an autotroph of capable growing with CO2 as a sole carbon source [Nature COpathways –8. Propiolecules . Methanon source . Hydroxy3) [This study focuses on the production of 2-hydroxyterephthalic acid (2-HTA), the monomer for high-performance poly(hydroxy-p-phenylenebenzobisoxazole) (HPBO) fiber. It is known that poly(p-phenylene benzobisoxazole) (PBO) fiber has excellent thermal stability (up to 650 °C), outstanding mechanical properties , low density (1.56g/cm3) , and has3) , 13. Bes3)  and meta3) , 16.2CO3/CO2 (atmospheric) and potassium formate solvent [Pseudomonas mutant was screened to oxidize Terephthalic acid (TPA) to a diol intermediate, 1,2-dihydroxycycclohexa-3,5-diene-1,4-dicarboxylic acid, and then the diol intermediate was dehydrated by sulfuric acid to form 2-HTA [ortho- and para-positions and requires high temperature. However, biosynthesis of 2-HTA has not been reported.Traditionally, 2-HTA is synthesized by chemical synthesis, including 2-bromoterephthalic acid (BTA) catalytic hydrolysis, and copper powder catalyzes BTA to generate 2-HTA at 100 °C in an alkaline environment; Koble–Schmitt method, 3-hydroxybenzoic acid (3-HBA) is carboxylated to 2-HTA at 230–240 ℃ in the presence of K solvent . Biosyntrm 2-HTA . The cheSaccharomyces cerevisiae. By culturing the recombinant strain in the CO2 gas, the de novo synthesis of 2-HTA based on the shikimic acid pathway is achieved using glucose as a raw material.In this study, we use the enzymatic Kolbe–Schmitt reaction to convert 3-HBA to 2-HTA. It is the first report to produce 2-HTA with these (De)carboxylases. Due to the low carboxylation activity towards 3-HBA, mutants obtained by site-directed mutagenesis are used to enhance catalytic activity. The chorismate lyase (XanB2) and 3-hydroxybenzoate synthase (Hyg5) have been reported to use chorismate as a precursor to synthesize 3-HBA , 19. Bot2 fixation. The family members including 2,6-dihydroxybenzoate decarboxylase  from Rhizobium sp. , 2,3-dihydroxybenzoic acid decarboxylase  from Aspergillus oryzae  [Fusarium oxysporum,  [Trichosporon moniliiforme (SAD_Tm) [ortho-position of the aromatic phenolic group. 2,6-DHBD_Rs has a narrow substrate spectrum and has no activity on monohydric phenols, such as phenol. 2,3-DHBD_Ao and SAD_Tm have broad substrate spectrum [para-position of aromatic carboxylic acids to form terephthalic acid derivatives. We use 3-HBA as a substrate to generate 2-HTA.Hydroxybenzoic acid (de)carboxylases is a reversible nonoxidative decarboxylases which has been used in Kolbe–Schmitt reaction with CODHBD_Ao) , 2,3-DHBDHBD_Fo) , 22, sal(SAD_Tm) , 24 showspectrum , only reE. coli BL21(DE3) whole cells expressing hydroxybenzoic acid (de)carboxylases catalyzed the natural substrate resorcinol to generate 2,4-dihydrobenzoic acid and 2,6-dihydrobenzoic acid in vitro, in which 2, 6-dihydrobenzoic acid was more than 2, 4-dihydrobenzoic acid. 2,3-DHBD showed better activity to resorcinol than SAD_Tm carboxylases from microbial sources have a very similar structure. It exists as a homotetramer, each subunit is a (β/α)8 barrel, the active site has a divalent metal ion, and the reaction mechanism is similar –30. The 3 was used in the enzyme carboxylation reaction in vitro. To verify whether the strain could grow normally in the KHCO3 solution, KHCO3 tolerance experiments were carried out on different strains.In general, saturated KHCOE. coli strains BL-0 and BL-A were used in the tolerance experiment. OD600 of BL-A first increased and then decreased in 0.5 M KHCO3 LB medium, at last remained at about 0.6. The OD600 of BL-0 decreased continuously in 1 M and 0.5 M KHCO3 LB medium. These proved that 1 M and 0.5 M KHCO3 inhibited the growth of E. coli (see Table .Saccharomyces cerevisiae strains BY-4741 (BY-01 and BY-A) were cultured in SC medium with 1 M and 0.5 M KHCO3. BY-01 and BY-A have an initial OD600 of 0.15. In 1 M and 0.5 M KHCO3, the growth of strains was inhibited, and OD600 was between 0.10 and 0.17 , the selectivity of 2,6-dihydroxybenzoic acid decreased as the concentration of KHCO3 declined. The selectivity was only 13.7% when the KHCO3 concentration dropped to 0.5 M. No 2,6-dihydroxybenzoic acid was produced, when the concentration of KHCO3 dropped to 0.1 M  and 17 (group 2) decreased sharply in the production of 2-HTA in the 0.3 M KHCO3. Compared with 0.5 M KHCO3, 2-HTA of group 1 decreased by 77.46%, and group 2 decreased by 79.55%  is used to replace KHCO3 for enzyme-catalyzed carboxylation. The pressure of CO2 has a great influence on carboxylation efficiency. In the literature, resorcinol was catalyzed by 2,3-DHBD_Ao , and the conversion reached 68%. As the CO2 pressure decreased, the conversion rate also decreased. Especially when the pressure was less than 20 bar, the conversion rate decreased sharply. At 10 bar, the conversion rate was very low, less than 20% [In the enzymatic carboxylation reaction, not only can KHCOthan 20% .In addition, pH is also an important parameter affecting the reaction. The stability of 2,3-DHBD_Ao and 2,6-DHBD_Rs is affected by pH. These two enzymes are active between pH 4–9, and the thermal stability is the highest between pH 6–7. In a low pH environment, the denaturation temperature decreases. Below pH 4.6, the enzyme is unstable at room temperature.3 will reduce the carboxylation efficiency, while a high concentration of KHCO3 will inhibit the growth of the strain. Therefore, CO2 is intended to replace KHCO3 as the carboxylation reagent in this experiment.The previous experiments have proved that the low concentration of KHCOS. cerevisiae converts one glucose molecule to two molecules of CO2 in the fermentation process that is beneficial to form the CO2 environment required for carboxylation. S. cerevisiae was chosen for the experiment. Hyg5 and (de)carboxylase were introduced exogenously, combined with the strain's shikimate pathway to construct a 2-HTA production pathway  and ordinary air environment (O2 group) were compared.The 2 group, OD600 reached 14. Ethanol reached the highest value of 12 g/L at 48 h, and then gradually decreased to 2.5 g/L at 96 h. Glycerol was ultimately metabolized without accumulation. In the CO2 group, OD600 was only 6. Ethanol accumulated to a concentration 20 g/L, and glycerol to 0.9 g/L. In these two groups, only small amounts of acetic acid were detected  was used as chassis strain. The strains SC-01, SC-H, SC-HA, SC-HA1 and SC-HA2  were cultured in 30 mL SC medium (0.05 M KHCO3). The initial OD600 was about 0.2, and the final OD600 was maintained at 5. The 3-HBA yields of SC-HA, SC-HA1 and SC-HA2 were 398.59 ± 28.43 mg/L, 366.94 ± 6.46 mg/L, and 352.69 ± 1.67 mg/L, respectively. The SC-HA produced 22.10 ± 1.56 μg/L of 2-HTA, which was close to that of 5D-HA, and the production of 2-HTA in SC-HA1 increased to 45.40 ± 0.28 μg/L carboxylases can catalyze the reversible decarboxylation reaction that converts phenols to hydroxybenzoic acids. In this study, the substrate spectrum of these enzymes was broadened, and the enzyme 2,3-DHBD_Ao was successfully used to generate the 2-HTA, the monomer for high-performance PHBO fiber. The enzyme activity was improved by site-directed mutagenesis. In vitro, whole-cell catalysts with the single point mutation F27G and T62A increased the production of 2-HTA by 6.3-fold and 8.2-fold. The double site mutation F27G/T62A increased the production of 2-HTA by 24.7-fold up to 2.69 ± 0.029 mg/L/mg protein.T62A were co-expressed in S. cerevisiae S288C which was knocked out of Ura3, Aro7 and Trp3. The engineered strain synthesized 2-HTA de novo in CO2 with a titer of 45.40 ± 0.28 μg/L.Combined with the shikimate pathway, a de novo biosynthesis pathway of 2-HTA was designed, from glucose to shikimate, chorismate, 3-HBA, and finally 2-HTA. 3-hydroxybenzoate synthase Hyg5 and hydroxybenzoic acid (de)carboxylases 2,3-DHBD_AoThis experiment tried to use bicarbonate as a carboxylation reagent and found that living cells can only tolerate low concentrations of bicarbonate. The sensitivity is not caused by bicarbonate-induced changes in solution pH, but may be related to bicarbonate regulation in biological systems.This pathway can be further developed on the productivity of TPA and its derivatives and the efficiency of carboxylases. It is also needed further research on intracellular utilization of carbon source.E. coli trans10 was used for propagating and storing the plasmids. E. coli BL21(DE3) was used for protein expression. S. cerevisiae strains were used for 2-HTA biosynthesis.The strains and plasmids used in this study were listed in Additional file E. coli strains were cultured in Luria–Bertani (LB) broth . The yeast strains were cultivated in synthetic complete (SC) medium without the auxotrophic compound complemented by the plasmids, which consisted of 6.7 g/L yeast nitrogen base (YNB), 5 g/L (NH4)2SO4, 0.77 g/L complete supplement mixture (CSM) without uracil or histidine, 20 g/L glucose.2,3-DHBD_Ao (NCBI reference sequence: XM_001817461.3) encoding 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae , 2,3-DHBD_Fo (GenBank: EXM04459.1) encoding 2,3-DHBD from Fusarium oxysporum , SAD_Tm (GenBank: DM040453.1) encoding salicylic acid decarboxylase from Trichosporon moniliiforme (SAD_Tm) and Hyg5 (GenBank: AF007101.1) encoding 3-hydroxybenzoate synthase from Streptomyces hygroscopicus, were synthesized by BGI Beijing (China), and cloned into appropriate vectors.Molecular cloning was performed according to standard protocols . The oli2,3-DHBD_Ao, mutants were amplified from pET28a( +)-2,3-DHBD_Ao using the corresponding up and down primers, and assembled by Gibson assembly [To construct site-directed mutation of assembly .E. coli BL21(DE3). E. coli BL21(DE3) strain was grown in LB medium at 37 ℃ and induced with 0.2 mM IPTG at OD600 1.0.2,3-DHBD was expressed in E. coli BL21(DE3) whole cells were suspended in 1 mL deionized water and rehydrated for 30 min, followed by adding substrate, 3 M KHCO3. The mixture was incubated at 30 ℃ for 72 h in sealed glass vials.In the whole-cell carboxylation reactions, 30 mg lyophilized The reaction mixture was stopped by adding 1 mL 6 M HCl. The resulting mixture was centrifuged at 12,000 rpm for 5 min. After dilution and filtration, the sample was analyzed by HPLC and HPLC–MS.2O/trifluoroacetic acid (0.1%) as the mobile phase (flow rate 0.8 mL/min) and an acetonitrile/trifluoroacetic acid (0.1%) gradient . Dionex/Thermo Ultimate 3000 system with an oscillometric detector was used to detect the concentration of glucose, ethanol, glycerol, and acetic acid concentration in the medium. Bio-Rad 87H column was used with 5 mM sulfuric acid as the mobile phase, a column temperature of 65 °C, and a flow rate of 0.6 mL/min.HPLC analysis was performed on Dionex/Thermo Ultimate 3000 system with a UV detector at 254 nm and a Hypersil GOLD C18 . The method was run over 18 min with H2O/formic acid (0.1%) as the mobile phase (flow rate 0.2 mL/min) and a methanol gradient . MS was operated in the negative-ion mode with electrospray ionization source (ESI).HPLC–MS/MS analysis was performed on the Shimadzu LC 20A system with SCIEX QTRAP 5500 systems as mass-detector and Agilent Pursuit PFP . The method was run over 15min with H600.Prepare LB culture medium with 0.1 M pH 6 potassium phosphate buffer instead of water. The strain BL-A expressing 2,3-DHBD_Ao and BL-0 strain expressing pET-28a( +) empty plasmid was inoculated into the LB culture medium containing 5 mM substrate 3-HBA. 1‰ IPTG was used to induce protein expression, and samples were taken at 0, 17, 35 h to measure OD3 was added into 100 mL anaerobic bottles and infused CO2 for 5 min. S. cerevisiae strains were inoculated and cultured at 200 rpm/min for 72 h.30 mL SC medium (30 g/L glucose) containing 1 M and 0.5 M KHCO2-saturated SC medium lacking the auxotrophic compound complemented by the plasmids. The metabolites were detected by HPLC and HPLC–MS/MS.For 2-HTA production, strains were cultured in the 100 mL anaerobic bottle at 30 ℃ and 200 rpm/min with 30 mL COAdditional file 1: Table S1. Enzymic carboxylation with natural substrates. Table S2. Quantitative analysis of protein expression in 2,3-DHBD—Ao, Aoopt, Fo, Foopt, Ao F27/T62. Table S3. Codon-optimized DNA sequences. Table S4. Strains and plasmids used in this study. Table S5. Primers used in this study. Figure S1. SDS–PAGE of purified 2,3-DHBD. Figure S2. 2-HTA production by 2,3-DHBD_Ao mutants with single and double amino acid substitutions in the pure enzyme catalysis. aReaction conditions: 10 mM substrate, 1mg/mL protein, 3 M KHCO3, 5 mM MgCl2, 30 ℃, 200 rpm.


Lactiplantibacillus plantarum WCFS1 possess two metabolicpathways for lactose metabolism, involving β-galactosidase and 6Pβ-galactosidase  activities. As L. plantarum WCFS1 genome lacks a putative 6Pβ-galgene, the 11 GH1 family proteins, in which their 6Pβ-glucosidase(6Pβ-glc) activity was experimentally demonstrated,, were assayedfor 6Pβ-gal activity. Among them, only Lp_3525 (Pbg9) also exhibiteda high 6Pβ-gal activity. The sequence comparison of this dual6Pβ-gal/6Pβ-glc GH1 protein to previously described dualGH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pβ-gal/6Pβ-glcGH1 proteins, as it possessed conserved residues and structural motifsmainly present in 6Pβ-glc GH1 proteins. Finally, Lp_3525 exhibited,under intestinal conditions, an adequate 6Pβ-gal activity withpossible relevance for lactose maldigestion management.Intestinal lacticacid bacteria can help alleviate lactose maldigestionby promoting lactose hydrolysis in the small intestine. This studyshows that protein extracts from probiotic bacterium  Individualswho suffer from lactose maldigestion must reduce lactose in theirdiet and avoid milk consumption. A conventional strategy for lactosemaldigestion management is to use milk products with reduced lactosecontent or to supplement the diet with exogenous β-gal. However,oral administration of β-gal is inconvenient and ineffectivebecause the exogenous enzyme has normally poor pH stability and israpidly degraded by proteases in the gastrointestinal tract.1 Two different metabolic pathwaysfor the lactose uptake have been reported in lactic acid bacteria.One pathway involves the translocation of intact lactose and furtherhydrolysis by a β-gal. In the other pathway, lactose is transportedinto cells via a lactose-specific phosphoenolpyruvate-dependent-phosphotransferasesystem (PEP–PTS), in which lactose is phosphorylated duringtransport and then hydrolyzed into 6-phospho-β-d-galactoseand d-glucose by 6P-β-galactosidase .4β-gal plays an important role inthe management of lactoseintolerance. Because β-galactosidases are also produced by gutmicrobiota, intestinal lactic acid bacteria can help alleviate lactosemaldigestion. Lactic acid bacteria that can reach and inhabit theintestinal tract may alleviate clinical symptoms brought about byundigested lactose and could promote lactose hydrolysis in the smallintestine.Lactiplantibacillus plantarum isone of the few lactic acid bacteria species that has successfullyadapted to different foods and is also part of the human colonic microbiota.7L. plantarum possess both metabolicpathways for lactose metabolism, as demonstrated by the fact thatcell-free extracts from several strains exhibited β-gal and6Pβ-gal activities.11 Several glycosidases exhibiting β-gal activityhave been described in L. plantarum strains, such as LacA13 and LacLM.17 However, the glycosidase(s) responsible for the 6Pβ-gal activityfound in L. plantarum cell extractsremains unknown.L. plantarum WCFS1 strain becauseit was isolatedfrom human saliva and persists in the digestive tract better thanother Lactobacillus spp. isolated fromthe human intestine.18 In this study, 6Pβ-galactivity was assayed in 11 glycoside hydrolases from L. plantarum WCFS1 possessing 6P-β-glucosidase(6Pβ-glc) activity. One of these proteins exhibited also a high6Pβ-gal activity and it was subsequently characterized to betterelucidate its dual 6Pβ-glc/6Pβ-gal glycosidase activity.This work aimed to study 6Pβ-gal activityin the L. plantarum WCFS1 strain used in this study waskindly provided by Dr. Michiel Kleerebezem . This strain is a colony isolate of L.plantarum NCIMB 8826, which was isolated from humansaliva. L. plantarum WCFS1 was grownin the modified MRS broth  at 30 °C. The MRS mediumwas modified by replacing glucose with galactose to avoid possiblecatabolite repression by glucose.19Escherichiacoli DH10B,used for DNA manipulations, and E. coli BL21(DE3), used for protein expression, were cultured in the Luria–Bertani(LB) medium containing ampicillin (100 μg/mL) at 37 °Cand 140 rpm.The L. plantarum WCFS1 were used to determine β-gal and 6Pβ-gal activitiesby using as substrates synthetic p-nitrophenyl-glucosidederivatives, pNP-β-d-galactopyranoside (Sigma-Aldrich) and pNP-6-phospho-β-d-galactopyranoside  (GoldBio),respectively.Restingcells and cell-free protein extracts from 20 Briefly,for the resting cell assays, L. plantarum WCFS1 cells were cultured in the MRS-Gal medium at 30 °C until0.5 OD600nm was reached. Then, cells were harvested andwashed twice with 0.9% w/v NaCl. After washing, cells were resuspendedin 50 mM MOPS buffer (pH 7.0) containing 20 mM NaCl and 1 mM DTT,and the corresponding p-nitrophenyl-glycoside (10mM) was added. Reactions were incubated at 30 °C for 10 min.Resting cells and cell-free protein extracts wereprepared as previously described.L. plantarum WCFS1 was grown in the MRS-Gal medium at 30 °C, until lateexponential phase. Cells were harvested, washed twice with 50 mM MOPSbuffer, NaCl 20 mM, 1 mM DTT, pH 7.0, and subsequently resuspendedin the same buffer for cell rupture. Bacterial cells were disintegratedby using a French Press and subsequently, cells were additionally broken with glass beadsin FastPrep TM Fp120 equipment (Savant). The disintegrated cell suspensionwas centrifuged at 4 °C in order to remove cell debris. Supernatantscontaining soluble proteins were filtered, and the obtained proteinextracts were incubated in the presence of the corresponding substrate(10 mM) at 30 °C for 10 min.For cell-free protein extracts, 20 The rate of hydrolysis of p-nitrophenyl (pNP) glycosides for 10 minat 30 °C was measured in 50 mM MOPS buffer pH 7.0 containing20 mM NaCl and 1 mM DTT, at 420 nm in a microplate spectrophotometerPowerWave HT . Reactions were stopped by the additionof 1 M sodium carbonate (pH 9.0). Control reactions containing noenzyme were utilized to detect any spontaneous hydrolysis of the substratestested. Enzyme assays were performed in triplicate.β-gal and 6Pβ-galactivities in resting cells and cell-freeprotein extracts were determined by a spectrophotometric method previouslydescribed.L.plantarum WCFS1 were produced as previously described.20 The hydrolytic activity of these 12 glycoside hydrolases was determinedby using a library of 24 pNP-glycoside derivatives.13 The assay was performed in a 96-well flat bottomplate (Sarstedt), where each well contained a different substrate(10 mM). Briefly, the reaction consisted of 4 μg protein in50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT. The reactionwas incubated at 30 °C for 10 min and stopped by the additionof 1 M sodium carbonate at pH 9.0. Hydrolysis of each pNP-glycoside derivative was colorimetrically measured by liberationof p-nitrophenolate (pNP) at 420nm in a microplate spectrophotometer PowerWave HT .Controls without the enzyme for evaluating spontaneous hydrolysisof the tested substrates were carried out. Experiments were performedin triplicate, and results were expressed as an average activities± standard deviation.Glycosidehydrolases from the GH1 family  and GH42 family (LacA or Lp_3469) from pNP-6-phospho-β-d-glucopyranoside (pNP-6P-Glc) and pNP-6-phospho-β-d-galactopyranoside  as substrates, for 6Pβ-glc and 6Pβ-gal activities,respectively. The optimal pH was determined by using citrate (pH 3.0),acetic acid-sodium acetate (pH 4.0–6.0), MOPS (pH 6.5 and 7.0),and Tris–HCl (pH 8.0) buffers (50 mM). The optimal temperaturewas assayed by incubation of Lp_3525 in 50 mM MOPS buffer pH 7.0 containing20 mM NaCl and 1 mM DTT at 4, 22, 30, 37, 45, and 65 °C. Assayswere performed in triplicate.The effects of temperature and pH on Lp_3525 weredetermined using L. plantarum OSU in asurvey of lactobacilli for β-galactosidase and phospho-β-galactosidaseactivities using pNP-galactoside derivatives as substrates.8 Later, using the same substrates, both activitieswere also identified in additional L. plantarum strains, extracts,10 or toluene-treatedcell suspensions.11 However, none of theseassays evaluated the activity exhibited by L. plantarum whole cells. When the lactose uptake occurs through the PEP-PTS,whole cells should not exhibit 6Pβ-gal activity because lactoseneeds to be phosphorylated during its transport into the cell. Inorder to know whether β-gal and 6Pβ-gal activities existin the probiotic L. plantarum WCFS1strain, both activities were assayed in resting cells and in cell-freeprotein extracts by using pNP-Gal and pNP-6P-Gal as substrates. As expected, resting cells did not show6Pβ-gal activity, and they exhibited only a low β-galactivity (0.47 ± 0.14 μmol min–1 mg–1). By contrast, both activities were observed at thesame extent in cell-free extracts, β-gal (3.67 ± 0.08 μmolmin–1 mg–1) and 6Pβ-galactivity (3.69 ± 0.06 μmol min–1 mg–1). This is the first report describing similar β-galand 6Pβ-gal activities in L. plantarum cell-free extracts. Previously, higher β-gal than 6Pβ-galactivities were reported in L. plantarum strains,11 except for L. plantarum ATCC 14917 and L. plantarum ATCC 8014 strains, which exhibited higher 6Pβ-gal activity.11 As β-gal activity was generally the predominantactivity in the previous reports, it was speculated that 6Pβ-galcould be a spurious enzymatic activity in L. plantarum.15Twodifferent pathways for lactose hydrolysis have been described in lacticacid bacteria, involving proteins exhibiting β-gal and 6Pβ-galactivities. These two activities were reported in crude cell-freeextracts from L. plantarum WCFS1 cell-free extracts was clearly confirmed, the proteins involvedin both activities should be identified. The available complete genomesequence of L. plantarum WCFS1 revealedthe existence of a high number of proteins annotated as “glycosidehydrolases”. Among the families included in the carbohydrate-activeenzyme (CAZy) database that contain proteins possessing β-galactivity, only GH1, GH2, and GH42 families are present in L. plantarum WCFS1. The presence of proteins annotatedas β-gal belonging to the families GH1 (Lp_3629), GH2 (Lp_3483and Lp_3484), and GH42 (Lp_3469) are found in the genome of L. plantarum WCFS1. L. plantarum β-gal from GH2 (LacLM) and GH42 (LacA) families have beenpreviously recombinantly produced and biochemically characterized.16 Both β-gal possess high transgalactosylation activity forthe synthesis of prebiotic galacto-oligosaccharides or other galactosylatedderivatives.16 In relation to GH1 family β-gal, it was experimentally proventhat Lp_3629 possesses β-gal activity, and not β-glc activityas it was originally annotated.21 Whenimmobilized, this β-gal successfully catalyzed the hydrolysisof lactose and lactulose, and the formation of different oligosaccharidesfrom galactose and lactulose was detected.22Once the presence of β-galand 6Pβ-gal activities in L. plantarum WCFS1 possesses11 genes encoding for GH1 family proteins that are annotated as 6PB-glc;however, there is no protein annotated as putative 6Pβ-gal.These 11 GH1 proteins have been recently characterized. All of thempresented 6Pβ-glc activity, and in addition, 8 proteins exhibited6-phospho-β-d-thioglucosidase activity.20 Therefore, it is possible that some of theseGH1 proteins could also possess 6Pβ-gal activity, being responsiblefor the 6Pβ-gal activity exhibited by L. plantarum WCFS1 cell-free extracts.According to the CAZy database, 6Pβ-gal is categorizedasa GH1 family enzyme. A total of 21 different enzymatic activitiesare currently classified in the GH1 family. Some enzymes, however,have broad substrate specificity, meaning that they can utilize morethan one substrate and catalyze more than one reaction. The completegenome of 20 The hydrolyticactivity of these enzymes was assayed by using pNP-β-d-galactopyranoside  and pNP-6-phospho-β-d-galactopyranoside  as substrates, for β-gal and 6Pβ-gal activities,respectively. LacA GH42 proteins were used as the control for β-galactivity.13 In addition, the catalyticactivity of these proteins toward 24 pNP glucosidederivatives was also assayed. Despite all the GH1 proteins exhibiting6Pβ-glc activity,20 only one of them,Lp_3525 (Pbg9), effectively hydrolyzed pNP-6P-Galand showed high 6Pβ-gal activity .The 11 GH1 6Pβ-glc proteinsand LacA β-gal were recombinantlyproduced as previously described.activity 1. Low 6Pactivity 1.13 NoneThe obtained results indicatedthat Lp_3525 has broad specificityas it could hydrolyze more than one phosphorylated substrate.23 Galacto- and gluco-derived substrates are very similar and haveidentical structures, differing only in the position of their O4 hydroxylgroup, which is an axial position in the galacto-epimer versus anequatorial position in the gluco-epimer. It was hypothesized thatboth enzymes evolved from a common ancestor, but at some point, 6Pβ-galevolved independently, so that a tryptophan residue was acquired inorder to accommodate galactose-6′P (rather than glucose 6-phosphate)at the active site of subsite −1.23Comparativestructural analysis suggests that a tryptophan instead of a methionineor alanine residue at subsite −1 may contribute to the catalyticand substrate selectivity to structurally similar 6Pβ-gal and6Pβ-glc assigned to the GH1 family.Geobacillus stearothermophilus is known.25 Gan1D has been shown to exhibitbifunctional activity possessing both 6Pβ-gal and 6Pβ-glcactivities. The different ligands trapped in the active site adoptdifferent binding modes to the protein, providing a structural basisfor the dual gal/glc activity observed for Gan1D enzymes. In thisprotein, specific mutations were performed on one of the active siteresidues (Trp-433), shifting the enzyme specificity from dual activityto a significant preference toward 6Pβ-glc activity from Lactococcus lactis subsp. lactis, and 4GPN (Bgl) from Streptococcusmutans as protein models for each specific activity,respectively (BlBglC numbering)involved in the hydrogen bond are not conserved in Lp_3525. Instead,Gly-426 and Val-431 residues (Lp_3525 numbering) are found. Theseresidues are conserved in the 6Pβ-glc 4GPN from S. mutans  or in the previously described dual 6Pβ-gal/6Pβ-glcprotein (Trp-433 in BlBglC) suggested that Lp_3525belongs to a different dual 6Pβ-gal/6Pβ-glc protein group, the residues involved in glyconbinding (−1 subsite), and those involved in aglycon binding(+1 subsite) are conserved.20 Moreover,the Lp_3525 sequence presents a long extra C-terminal segment uniqueto 6Pβ-glc, which varies in length and sequence. This is a typical6Pβ-glc motif that is absent in the 6Pβ-gal hydrolases.29 In addition, Lp_3525 lacks the lid motif presentin 6Pβ-gal that blocks the entrance to the active site instead of the asparagine and tryptophan present in the 6Pβ-gal(Asn-297 and Trp-429 in 4PBG)  thanto 4PBG 6Pβ-gal (35.76% identity). All these data seem to indicatethat Lp_3525 belongs to a dual 6Pβ-gal/6Pβ-glc enzymegroup but more closely resembling the structural features of 6Pβ-glcproteins.The sequence of Lp_3525 has typicalresidues conserved in 6Pβ-glc.ive site 1.29 Lp_3in 4PBG) 1.30 All BlBglC, GK3214, and Gan1Dproteins havebeen previously described as “dual 6Pβ-gal/6Pβ-glc”,28 despite presenting a similarsequence identity to 4GPN and 4PBG .Moreover, they possess residues and motifs specific for 6Pβ-gal,such as the asparagine and tryptophan residues (Asn-297 and Trp-429in 4PBG),30 and the lid motif that blocks the entranceto the active site.29 Additionally, thelong extra C-terminal segment typical of 6Pβ-glc is absent inBlBglC, GK3214, and Gan1D proteins. These data indicated, that contrarilyto Lp_3525, these dual 6Pβ-gal/6Pβ-glc are more similarto 6Pβ-gal enzymes.However, Lactobacillus gasseri ATCC33323, four enzymes showing6Pβ-gal activity  were described.Phylogenetic analysis showed that LacG1 and LacG2 belongs to the 6Pβ-galcluster and Pbg1 and Pbg2 belongs to the 6Pβ-glc cluster, asthey showed closer similarity to 6Pβ-glc (about 50%) than to6Pβ-gal (30–35%).31 The four L. gasseri GH1 glycosidases exhibited dual 6Pβ-gal/6Pβ-glcactivity, as they were able to hydrolyze oNP-6P-Galand oNP-6P-Gal.32 As inthe case of Lp_3525, Pbg1 and Pbg2  showed all the residues and motifs conservedin 6Pβ-glc .28 However, gathered evidenceindicates that there is a different group of GH1 glycosidases possessingdual 6Pβ-gal/6Pβ-glc activity whose the structural basisfor their enzyme bifunctionality remains unknown.Therefore, so far, the differences in gluco and galactosubstrate-bindinginteractions in dual 6Pβ-gal/6Pβ-glc GH1 glycosidaseshave only been analyzed in proteins sharing sequence and structuralfeatures with 6Pβ-gal  assayed with pNP-6P-Glc  and LacG2 (40 °C) from L. gasseri for both substrates.32 In addition, Lp_3525 possessed a different optimal pH foreach enzymatic activities, being more acidic for 6Pβ-gal (pH4.0) than for 6Pβ-glc (pH 6) activity 34 to 50 °C (LacG from S. cremoris and Pbg1 from L. gasseri JCM1032).35 Most of these 6Pβ-gal were inactivated above 60 °C, asit happened in Lp_3525 whose optimal temperature was 45 °C 36 or 2% at60 °C (CelD)37 whereas Lp_3525 retainedat 65 °C more than 95% of its maximal activity 36 or 25% at pH 7.5 (CelD).37When Lp_3525 6Pβ-glcactivity was compared to other 6Pβ-glcpreviously described, it was observed that Lp_3525 possessed a broadrange of optimal temperature and pH 2. Lp_352activity 2b. As meL. gasseri ATCC 33323), Lp_3525, which belongs tothe 6Pβ-glc group, possessed different biochemical propertiesconcerning the hydrolyzed substrate, pNP-6P-Gal or pNP-6P-Glc. Moreover, Lp_3525 exhibited a broad range oftemperatures and pH for its maximal 6Pβ-glc activity, althoughit displayed more constricting conditions for 6Pβ-gal activity.The obtained data confirmed that similarlyto dual 6Pβ-gal/6Pβ-glcbelonging to the 6Pβ-gal group  butalso 6Pβ-glc. This strain contained seven different genes associatedwith 6Pβ-glycosidases, at least four of them encoded dual 6Pβ-gal/6Pβ-glcproteins. Pbg1 and Pbg2 belong to the 6Pβ-glc group describedin this study for the first time, whereas the primary enzymes forlactose utilization, LacG1 and LacG2, belong to the 6Pβ-galgroup previously described.32 In L.plantarum WCFS1, among the 11 proteins having 6Pβ-glcactivity,20 only Lp_3525 possessed high6Pβ-gal activity. Although it appears to be a waste of energythat several enzymes possess apparently the same function, resultspreviously obtained by using 6Pβ-glc mutants,43 and those described in this work confirmed that not allthe proteins are functionally complementary. It has been describedthat the gene encoding Lp_3525, the L. plantarum WCFS1 dual 6Pβ-gal/6Pβ-glc protein, is controlled bycarbon catabolite repression,44 but furtherstudies are needed in order to know the regulation and expressionof Lp_3525 in the presence of lactose and different carbon sources,and its possible relevance for lactose maldigestion management.45 Asa consequence, these enzymes could be useful in bioprocessing approachesfor the valorization of some cereal byproducts following their inclusionin lactic acid bacteria starters with targeted metabolic activities.46In addition to lactose metabolism, in this work, we have demonstratedthat Lp_3525 could be able to hydrolyze phospho-β-galacto/gluco-derivedsubstrates over a broad temperature and pH ranges, especially forthe 6Pβ-glc activity. Although the capability of phospho-β-glycosidaseactivities is still rather unexplored, this type of enzymes may catalyzethe degradation of phosphorylated glucosides, glycosylated lignin,and fiber-related disaccharides during plant-based fermentations and contribute to the release ofa wide range of phenolic compounds.


In the crystal, the non-H atoms of the complex are practically coplanar (r.m.s. deviation of fitted atoms = 0.135 Å), and the angle between the thienyl and the chelating rings is 6.7 (1)°. The molecules stack at a distance of 3.623 (2) Å along the b-axis direction.In the title complex, [Ni(C These values agree with those reported in previous papers  atom is located on a crystallographic center of symmetry and exhibits a square-planar coordination sphere, being coordinated by two negatively charged N,S-chelating ligands in a All of the non-H atoms of the complex are almost coplanar, with S1 and C1 [−0.28 Å] and C13, C14 , respectively deviating the most from its mean plane (r.m.s. deviation of fitted atoms = 0.135 Å). The thienyl ring forms a small dihedral angle of 6.7 (1)° with respect to the chelating five-membered ring. The long alkyl chain is in a staggered conformation with torsion angles along the chain that range between 176.7 (2) and 179.8 (2)°.x, 1 − y, 1 − z] of the symmetry-related ligand .The molecule is stabilized by an intramolecular unconventional hydrogen bond between C5—H5 with S1′ : 475, 400, 276.UV–Vis spectrum [CHCl28H42N4NiS6 [M+H]+: 685.11599, found [M+H]+: 685.11549.HRMS (FAB) Calculated for C6.Uiso(H) = 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement are summarized in Table 210.1107/S2056989023005935/pk2690sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989023005935/pk2690Isup2.hklStructure factors: contains datablock(s) I. DOI: 2254902CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the β-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides.  •Epoxy-enone (EE) ceramides form stable conjugates with Cys•Cys binds to the β-carbon of the enone via a Michael addition reaction•Cys-bound EE ceramides exist in the mouse epidermis•Cys-bound EE ceramides are the predominant protein-bound ceramides Abnormalities in skin barrier function increase the risk of or cause infectious diseases, atopic dermatitis, and ichthyosis, a skin disorder characterized by dry, thickened, and scaly skin.,O-acylceramides). The FA moiety of acylceramides is hydroxylated, and linoleic acid is attached to the ω-hydroxyl group via an ester linkage. This unique acylceramide structure, which contains a very long N-acyl moiety (C30–C36) and features additional O-acylation of the ω-hydroxyl group, forms and maintains the lipid lamellae.,,,,,,,,,,,,,,,,,,,,,,,,,,Ceramides are composed of a long-chain base (LCB) and an amide-linked FA. Numerous ceramide classes with different combinations of LCBs and FAs are found in mammalian epidermis.Two models have been proposed for the structure of protein-bound ceramides. In the first model, the ω-hydroxyl group of ceramides with ω-hydroxy (ω-OH) FA (ω-OH ceramides) is ester-linked to the Glu residues of CE proteins .ALOX12B and ALOXE3 are the causative genes of congenital ichthyosis.in vitro experiment,in vivo remains controversial.,P-O ceramides are considered to be produced via the following pathway. First, the linoleic acid moiety of acylceramides undergoes peroxidation and subsequent epoxy-hydroxylation by successive reactions catalyzed by the lipoxygenases ALOX12B and ALOXE3, respectively, producing epoxy-hydroxy ceramides .38 Both SDR9C7 converts epoxy-hydroxy ceramides to EE ceramides.,,The P-EO ceramide model was recently proposed based on the finding that the dehydrogenase encoded by the ichthyosis causative gene To date, no methods for detecting/quantifying P-O ceramides/P-EO ceramides in the peptide- or amino acid-bound state have yet been established due to technical difficulties. Instead, ω-OH ceramides released from P-O/P-EO ceramides by hydrolyzing the ester bond with alkali are measured. However, this method cannot discriminate between P-O ceramides and P-EO ceramides, leaving the presence and quantity of each unclear.In this study, we established a detection/quantification method for P-EO ceramides and revealed the target amino acid residue (Cys) and reaction mode (Michael addition reaction) of EE ceramides. Furthermore, we show that Cys-bound P-EO ceramides are the predominant protein-bound ceramides. These findings provide an important basis for elucidating the molecular mechanisms of skin barrier formation and for developing diagnosis and therapy for skin diseases caused by abnormal skin barrier formation.To examine whether EE ceramides bind to nucleophilic amino acids, we first used a chemically synthesized analog of EE ceramides  and the proton adduct of EE-analog, respectively. We detected a molecular ion peak of EE-analog–Cys when the EE-analog was incubated with Cys  coupled MS by selecting molecular ions with with Cys D. Moreovwith Cys E. Anothewith Cys D, it sho1H NMR analysis was performed. The signals corresponding to the olefinic protons of an α,β-unsaturated ketone  and enzymatic (cleavage with epoxide hydrolase EPHX2) reactions, respectively. TLC analysis confirmed high purity of the synthesized compounds  in which the keto group of EE-analog was reduced; a dihydroxy form  with a cleaved epoxy group; and an α,β-unsaturated carbonyl compound (2-octen-4-one) without an epoxy group A. Exceptompounds A. Epoxy- analogs B.Figure Each compound was incubated with Cys, and the reaction products were separated by TLC and stained with copper phosphate and ninhydrin. Cys conjugates of EE-analog, dihydroxy-enone, and 2-octen-4-one, all of which contain enone  structures, were detected by both detection methods B. Converm/z = 264) detected molecular ions ([M + H]+ and [M + H–H2O]+) corresponding to the EE ceramides containing saturated or monounsaturated ω-OH FAs with a C30 to C36 chain length   by long-term incubation with organic solvents.n length A and S3Cz = 309) B and S3Dm/z of 1,190, 1,192, 1,218, and 1,246  or with Cys, and the reaction products were subjected to precursor ion scan analysis to detect fragment ions with a sphingosine structure. The reaction product of EE ceramides alone produced ion peaks in the LC retention time range of 12.6 min–13.9 min, which corresponded to the molecular ion peaks of EE ceramides with C30 to C36 ω-OH FAs A and 4B,de alone A and S4Bde alone C.Figure + and [M + H–H2O]+ ions of EE ceramides or EE ceramide–Cys conjugates as the precursor ions and an ion with sphingosine structure (m/z = 264) as the product ion for MRM measurements. When EE ceramides and Cys were incubated, we detected EE ceramide–Cys conjugates with C30 to C36 saturated and monounsaturated ω-OH FAs  mode of LC-MS/MS. MRM mode can detect ions specifically and with high sensitivity by selecting both precursor and product ions. Here, we selected [M + H]ω-OH FAs C. The toω-OH FAs C and 4D.ω-OH FAs , with alω-OH FAs D. These in vitro binding assays between EE-analog or EE ceramides and amino acids were performed at pH 7.4. The pH of the skin surface is 4–6, with a higher pH in the deeper stratum corneum, and that of the interface between the stratum granulosum and the stratum corneum, where protein-bound ceramides are formed, is approximately 7.,The aforementioned Involucrin has been identified as the major binding target protein of P-O ceramides.m/z = 264) of LC-MS/MS. We detected two peaks (peaks 1 and 2) for the reaction products, which preceded the peaks of EE ceramides  and EE ceramides containing C32:1 and C34:1 ω-OH FAs, respectively . These results indicate that EE ceramides can bind at least to involucrin peptides.Next, EE ceramides were incubated with peptide 1, and the reaction products were subjected to precursor ion scan analysis  by LC-MS/MS. We detected Cys-bound P-EO ceramides with C30:0 , C32:1 , C32:0 , and C34:1  ω-OH FAs in wild type (WT) epidermis in a pronase E treatment-dependent manner , 1,271 (Arg), and 1,243 (Lys), and those of Schiff base products are 1,253 (Arg) and 1,225 (Lys).To examine whether Cys-bound P-EO ceramides are indeed present in epidermis, protein-bound ceramide fraction of mouse epidermis was treated with pronase E to degrade proteins to amino acids, followed by precursor ion scan analysis , few peaks were detected in these regions . We dete2O2) was added during the incubation of the protein-bound ceramide fraction with organic solvent (90% CH3OH). Elution of EE ceramides from the protein-bound ceramide fraction decreased in a DTT concentration-dependent manner , while addition of H2O2 increased elution  ..4-fold) D. These .4-fold) E.We performed quantitative analysis to determine the proportion of P-EO ceramides in protein-bound ceramides in epidermis. The protein-bound ceramide fraction , reversibly bound fraction (Cys-bound P-EO ceramides), and irreversibly bound fraction  were each converted to ω-OH ceramides by alkaline treatment, and the resulting ω-OH ceramides were quantified by LC-MS/MS A. When p,,Although the P-O ceramide model has long been considered the structure model of protein-bound ceramides,In the experiment that led to the development of the P-O ceramide model, peptide–protein-bound ceramide conjugates obtained from the protein-bound ceramide fraction of human foreskin by proteinase K treatment were analyzed via MS.in vitro conditions at least, the transglutaminase TGM1 exhibited this bond-forming activity.In vivo, protein-bound ceramide levels in the epidermis of Tgm1 KO mice were ∼60% of those in WT mice.In the predicted P-O ceramide synthesis pathway, a bond is formed between the ω-OH ceramides and CE proteins in the final step. Under in vitro.in vivo. For example, the epoxy group can bring EE ceramides into close proximity with Cys residues by interacting with polar groups of CE proteins. Alternatively, the epoxy group may itself promote reactivity between EE ceramides and the thiol group of Cys residues. The pKa of the thiol group of the Cys residue is 8.4 and the thiol group of the CE proteins is thus protonated and consequently not highly nucleophilic. However, it is possible that the unshared electron pair of the oxygen atom of the epoxy group attracts the thiol group proton, thereby enhancing thiol nucleophilicity and promoting the Michael addition reaction between the thiol group and the EE ceramides. The dihydroxy-enone analog also bound to Cys .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Akio Kihara . Mice at age of 3–12 months were used for breeding. Female and male mice at postnatal day 0 were used for the experiments. Healthy mice were used for the breeding and experiments. Animal experiments were approved by the institutional animal care and use committee of Hokkaido University and conducted in accordance with their guidelines.C57BL/6J mice were purchased from Sankyo Labo Service Corporation . 2 at 37°C. Transfection was performed using PEI Max  as described previously.HEK 293T cells were obtained from RIKEN BioResource Research Center  and cultured in Dulbecco’s Modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/mL penicillin, and 100 μg/mL streptomycin (Merck). Cells were grown on collagen coat dishes  and incubated under 5% COE)-oct-2-en-7-ynoate) was synthesized as below. 5-Hexyn-1-ol  was added dropwise to a mixture of Dess-Martin periodinane2Cl2 (45 mL) at 0°C over 20 min. The mixture was warmed to room temperature and stirred for 2.5 h. The reaction was quenched with a solution of sat. aq. NaHCO3 and sat. aq. Na2S2O3, after which the mixture was partitioned between CH2Cl2 and sat. aq. NaHCO3. The organic phase was washed with sat. aq. NaHCO3 and brine, then Na2SO4 was added to the organic phase to remove trace amounts of water. The mixture was filtered and concentrated in vacuo to afford a crude aldehyde. A mixture of the crude aldehyde and triethyl phosphonoacetate  was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene  at 0°C for 16 h. The mixture was partitioned between ethyl acetate and 1 M aq. HCl. The organic phase was washed with 1 M aq. HCl, sat. aq. NaHCO3 (×2), and brine, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was then purified by flash silica gel  column chromatography  to afford compound 1  as a colorless oil. 1H NMR  δ 6.94 , 5.86 , 4.18 , 2.33 , 2.23 , 1.97 , 1.69 , 1.29 . This is a known and reported compound.EE-analog was synthesized via a scheme composed of six reaction steps , where eE)-oct-2-en-7-yn-1-ol) was synthesized as below. A solution of compound 1  in CH2Cl2 (15 mL) was treated with diisobutylaluminium hydride  at −78°C for 3 h. The mixture was warmed to room temperature and stirred for 11 h. The reaction was quenched with sat. aq. NH4Cl at 0°C. After stirring the mixture at room temperature for several minutes, an insoluble material was filtered off through a Celite pad (Merck) and the filtrate partitioned between CH2Cl2 and brine. The organic phase was washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by flash silica gel column chromatography  to afford compound 2  as a colorless oil. 1H NMR  δ 5.68–5.67 , 4.10 , 2.31–2.15 , 1.96 , 1.62 ; m/z 124.9 [M + H]+.Compound 2 -3-(pent-4-yn-1-yl)oxiran-2-yl)methanol, was synthesized as below. A solution of compound 2  in CH2Cl2 was added to a suspension of diisopropyl D-tartrate , tetraisopropyl orthotitanate , and molecular sieves 4A  at −20°C. Then, tert-butyl hydroperoxide  was added dropwise over 1 min, and the mixture was stirred at −20°C for 2 h. The reaction was quenched with triphenylphosphine , then purified by flash silica gel column chromatography  to produce compound 3  as a colorless oil. 1H NMR  δ 3.91 , 3.64 , 2.98 , 2.95 , 2.28–2.25 , 1.97 , 1.80–1.64 ; m/z 163.2 [M+Na]+.Compound 3, ((2E)-1--3-(pent-4-yn-1-yl)oxiran-2-yl)oct-1-en-3-one) was synthesized as below. A mixture of compound 3  and NaHCO3  in CH2Cl2 (5 mL) was treated with Dess-Martin periodinane  at 0°C for 5 min. The mixture was warmed to room temperature and stirred for 16.5 h. The reaction was quenched with a solution of sat. aq. NaHCO3 and sat. aq. Na2S2O3, then the mixture was partitioned between ethyl acetate and sat. aq. NaHCO3. The organic phase was washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo to produce a crude aldehyde. A mixture of the crude aldehyde in CH2Cl2 was treated with compound 4 , which was synthesized as reported1H NMR  δ 6.51 , 6.39 , 3.24 , 2.92 , 2.52 , 2.27 , 1.97 , 1.86–1.66 , 1.61 , 1.37–1.25 , 0.89 ; m/z 235.1 [M + H]+.EE-analog  using primers  and cloned into the TA cloning vector pGEM-T Easy Vector . The EPHX2 fragment was excised with restriction enzymes SalI and NotI and cloned into the pCE-puro 3×FLAG-1, the mammalian expression vector to produce N-terminally 3×FLAG-tagged proteins,The plasmid to express epoxide hydrolase EPHX2 in mammalian cells was generated as follows. The coding sequence of g, 4°C, 3 min) and the total cell lysates thus prepared were subjected to ultracentrifugation . The resulting supernatant (soluble fraction) was recovered and used as an enzyme source of dihydroxy-enone-analog preparation, as described below.EPHX2-containing soluble fraction was prepared from HEK 293T cells overexpressing 3×FLAG-EPHX2 as follows. After 24 h from transfection of pCE-puro 3×FLAG-EPHX2 into HEK 293T cells, the cells were washed with PBS, harvested by a scraper, and transferred to a plastic tube. The cells were suspended in lysis buffer , and lysed by sonication. Unbroken cells were removed by centrifugation (400 × 3OH were incubated with 2 mg/mL NaBH4 (Merck) in 150 μL of CH3OH at 37°C for 1 h. Following the reactions, samples were mixed with 300 μL of CHCl3 and 270 μL of H2O and centrifugated . The lower, organic phase containing epoxy-enol-analog was recovered, dried, and dissolved in 750 μL of CH3OH.Epoxy-enol-analog was synthesized by reducing the EE-analog. 10 mM EE-analog in 150 μL of CH3/CH3OH = 1:2 (v/v), 250 μL of CHCl3, and 250 μL of H2O, successively. After centrifugation , the lower, organic phase containing dihydroxy-enone analog was recovered, dried, and dissolved in 100 μL of CH3OH.Dihydroxy-enone-analog was produced by cleaving the epoxy group using the soluble fraction prepared from HEK 293T cells overexpressing EPHX2, as described above. The mixture of 20 μL of 10 mM EE-analog and 180 μL of the soluble fraction (100 μg protein) was incubated at 37°C for 1 h. Samples were then mixed with 750 μL of CHCl3/CH3OH/acetic acid = 94:4:1 (v/v) as the solvent system. The plate was sprayed with copper phosphate solution (8% [v/v] aqueous phosphoric acid containing 3% [w/v] copper acetate), dried, and heated at 180°C for 5 min. The LC-MS analysis is described below.The structures of the produced compounds were confirmed by TLC and LC-MS analysis. EE-analog and its derivatives were spotted onto a TLC plate  and separated using CHCl3OH and 5 μL of 2 mM amino acid  in 50 mM Tris-HCl (pH 7.4) were incubated at 37°C for 1 h. The reaction products were spotted onto TLC plate and separated using 1-butanol/acetic acid/H2O = 3:1:1 (v/v) as the solvent system. The TLC plate was sprayed with copper phosphate solution or ninhydrin solution , dried, and heated at 180°C for 5 min (copper phosphate solution) or at 120°C for 5 min (ninhydrin staining). The binding assays using EE-analog derivatives and Cys, or EE-analog and peptides, were also conducted using the same procedure.The mixture of 5 μL of 2 mM EE-analog in CH3OH, 300 μL of CHCl3, and 265 μL of H2O, successively. After centrifugation , the lower, organic phase was collected, dried, dissolved in 1 mL CH3OH, and subjected to LC-MS analysis , as described below.The produced EE-analog–Cys conjugate was extracted for LC-MS analysis as follows. A portion of the above reaction product (10 μL) was mixed with 295 μL of CH3OD; Cambridge Isotope Laboratories) and 250 μL of 112 mM Cys (dissolved in D2O [Cambridge Isotope Laboratories] containing 112 mM triethylamine) was incubated at 25°C for 5 min and subjected to 1H NMR spectrometry at 400 MHz .The mixture of 250 μL of 44 mM EE-analog , and crushed  using a Micro Smash MS-100 (TOMY Seiko). The solutions were transferred to a glass tube and centrifugated , and supernatants were removed. The pellets were then treated with 2 mL of CHCl3/CH3OH , vigorously mixed, and centrifuged . To remove free lipids completely, this procedure was repeated five times, and the resulting pellets were recovered as protein-bound ceramide fractions.Skin was collected from mice sacrificed by decapitation at postnatal day 0 and incubated with 2 mg/mL Dispase II (Thermo Fisher Scientific) in PBS at 4°C for 16 h. The epidermis was separated from the dermis using forceps, washed with PBS, and weighed after removing excess water by pressing onto filter paper. The epidermis (5 mg) was transferred into a tube containing zirconia beads , suspended in 500 μL CHCl3/CH3OH  at 60°C for 3 h (first and second rounds) or for 16 h (third round) and centrifuged  to separate supernatants and pellets. The supernatants were collected, and 300 μL of CHCl3/CH3OH  was added to the pellets each time. The supernatants were then pooled, dried, dissolved in 500 μL of 90% CH3OH, and mixed with 500 μL of hexane. After centrifugation , the upper phase was collected. To the lower phase, 500 μL of hexane was added again and vigorously mixed. After centrifugation , the upper phase was collected. The two upper phases were combined, dried, and dissolved in CH3OH.EE ceramides were prepared by repeating the following procedure three times. The protein-bound ceramide fraction was incubated with 500 μL of CHCl3OH containing DTT or H2O2. After centrifugation , the supernatants were collected, mixed with 270 μL of CHCl3 and 213 μL of H2O, successively, and centrifuged again . The lower, organic phase was collected, dried, dissolved in 1.5 mL of CHCl3/CH3OH , and subjected to LC-MS/MS analysis  as descried below.In the experiment to determine whether the reversible release of EE ceramides was due to sulfoxide elimination, the protein-bound ceramide fractions (0.05 mg) were incubated at 60°C for 3 h with 300 μL of 90% CH3OH, after which 10 μL of the suspension (corresponding to 0.05 mg of the epidermis) was aliquoted. The following procedure was repeated five times to elute reversible protein-bound ceramides. Samples were incubated with 300 μL of CHCl3/CH3OH  at 60°C for 3 h  or for 16 h (third round), followed by centrifugation . The supernatants were collected and 300 μL of CHCl3/CH3OH  was freshly added to the pellets each time. The supernatant of each fraction was dried and used as reversibly bound ceramide fractions. The pellets derived after the five-time elution were used as irreversibly bound fractions. The protein-bound ceramide fractions, reversibly bound fractions, and irreversibly bound fractions were each suspended in 300 μL of 1 M KOH in 95% CH3OH containing 2 μL of 1 μM N-(2'-(R)-hydroxypalmitoyl(d9)) D-erythro-sphingosine  as an internal standard and incubated at 60°C for 1 h to release ω-OH ceramides from protein-bound ceramides. The samples were neutralized with 17.1 μL of 17.5 M acetic acid and mixed with 285 μL of CHCl3 and 239.4 μL of H2O successively. After centrifugation , the lower, organic phase was collected, dried, dissolved in 500 μL CHCl3/CH3OH , and subjected to LC-MS/MS analysis  as described below.The protein-bound ceramide fractions prepared from 5 mg of the epidermis as described above were suspended in 1 mL CH3OH and transferred into a plastic tube. After centrifugation , the pellets were washed with 500 μL of 50 mM Tris-HCl (pH 7.4), suspended in 135 μL of 50 mM Tris-HCl (pH 7.4) containing 1 mM DTT by sonication, treated with 15 μL of 10 mg/mL Pronase E , and incubated at 37°C for 2 h. The samples were then mixed with 562.5 μL CHCl3/CH3OH , 187.5 μL CHCl3, and 187.5 μL H2O, successively. After centrifugation , the lower, organic phase was collected, dried, dissolved in 50 μL of CHCl3/CH3OH, and subjected to LC-MS/MS analysis , as described below.Protein-bound ceramide fractions prepared from 10 mg of the epidermis as described above were suspended in 1 mL of CH3CN/H2O  containing 5 mM ammonium formate) and mobile phase B  at 55°C with a flow rate of 0.3 mL/min. EE-analog and EE-analog–Cys conjugate were separated using the following gradient conditions: 0 min, 0% B; 0–3 min, linear gradient to 10% B; 3–10 min, linear gradient to 40% B; 10–18 min, linear gradient to 100% B; 18–23 min, 100% B; 23 min, return to 0% B; 23–25 min, 0% B. EE ceramides and EE ceramide–Cys conjugates were separated using the following gradient conditions: 0 min, 40% B; 0–18 min, linear gradient to 100% B; 18–23 min, 100% B; 23 min, return to 40% B; and 23–25 min, 40% B. Ionization was performed by electrospray ionization and MS parameters were set as follows: capillary voltage, 2.5 kV; sampling cone, 30 V; source offset, 50 V; desolvation temperature, 650°C; desolvation gas flow, 1,200 L/h; cone gas flow, 150 L/h.EE ceramides, EE-analog, and their Cys adducts were detected using an ultra-performance LC-coupled with tandem triple quadrupole mass spectrometer . Separation by LC was performed using a reverse phase column  and a binary gradient system comprising mobile phase A  system and data analysis were performed using MassLynx software . Quantification of ω-OH ceramides was performed by calculating the ratio of the peak area for each ω-OH ceramide species to the internal standards.EE-analog and EE-analog–Cys conjugate were detected using a single ion monitoring mode, in which molecular ions with respectively t test in Microsoft Excel  and the significance of differences to controls in the presence of multiple groups was tested using Dunnett’s test in JMP13 . A p-value of <0.05 was considered significant.Quantification was performed as described in the


Both of the structures consist of a tridentate ligand synthesized from l-valine and salicylaldehyde, and one water molecule or two water molecules coordinating to CuII.The molecular structure of the title compound, [Cu(C 12H13N2O3)(H2O)2]·[Cu(C12H13N2O3)(H2O)], consists of two different molecules in the asymmetric unit. Both of the structures consist of a tridentate ligand synthesized from l-valine and salicylaldehyde, and one water molecule or two water molecules coordinating to CuII. They have a square-planar (molecule 1) or a square-pyramidal (molecule 2) coordination geometry. In the crystal, the molecules form intra- and intermolecular O—H⋯O hydrogen bonds involving the coordinated water molecules and other sites. A Hirshfeld surface analysis indicated that the most important contributions to the packing are from H⋯H [52.9% (molecule 1) and 51.1% (molecule 2)] and H⋯O/ O⋯H [21.2% (molecule 1) and 25.8% (molecule 2)] contacts. In addition, an electrostatic potential map was also obtained from DFT calculations to support the discussion of the intermolecular interactions.The molecular structure of the title compound, [Cu(C They play an important and diverse role in coordination chemistry  Å, which is close to the typical C=N double bond length for imines , is 2.6586 (17) Å.The asymmetric unit of the title compound consists of two different molecules, molecule 1 (containing atom Cu1) and molecule 2 (including Cu2). In molecule 1, Cu1 has square-planar geometry, being coordinated by a tridentate ligand synthesized from et al., 2020et al., 2020et al., 2020x, y, z) = 2.7937 (16) Å and Cu2—O9 = 2.3663 (16) Å; the latter is longer than Cu2—O6 because of the pseudo-Jahn–Teller effect. The Cu2—O6 bond is slightly longer than Cu1—O2 as a result of the crowding that occurs as the number of coordinating water molecules increases.In contrast, in molecule 2 the Cu2 atom exhibits a square-pyramidal geometry, being coordinated by the same tridentate ligand in the equatorial plane and by two water molecules in the equatorial and axial positions. The C19=N2 double-bond distance is 1.278 (3) Å, which is again close to the typical C=N double-bond length for imines —Cu1—N1 [102.50 (6)°] is tilted by much more than 90° as a result of the O9—H12⋯O4ii hydrogen bond. Similarly, the angle O8—Cu2—N2 [98.58 (6)°] is tilted by more than 90° because of the effect of the O2—H2B⋯O8 hydrogen bond. In the crystal, the molecules form an infinite chain as a result of the interaction of these six hydrogen bonds and the CuII atom with the carbonyl groups of the ligands  < 2.0 Å in the 2D fingerprint plots  and the calculation was performed using the The bond lengths and bond angles of the square-planar sites are generally consistent between the crystal structure and the optimized structure Table 2. In the 5.et al., 2016) for similar structures returned three relevant entries: (aqua-O)(methanol-O)[N-salieylidene-l-threoninato]copper(II) [N-salieylidene-l-threoninato]copper(II) [N-salieylidene-l-valinato]copper(II)  was reacted with salicylaldehyde  in methanol (20 mL), which was treated with microwave irradiation at 358 K for 5 min to yield a yellow ligand solution. Copper(II) acetate monohydrate  was added to the ligand solution and treated with microwave irradiation at 358 K for 5 min to yield a green solution. For recrystallization, the green solution was placed in the air at 300 K for several days, and the title complex was obtained  as green needle-shaped single crystals suitable for single-crystal X-ray diffraction experiments. Elementary analysis: found: C, 47.78; H, 5.12; N, 4.61%. Calculated: C24H32Cu2N2O9, C, 46.52; H, 5.21; N, 4.52%. IR (KBr): 1026 cm−1 (m), 1076 cm−1 (w), 1134 cm−1 (w), 1152 cm−1 (m), 1197 cm−1 (m), 1286 cm−1 (w), 1317 cm−1 (m), 1370 cm−1 (m), 1450 cm−1 , 1455 cm−1 (m), 1535 cm−1 (m), 1600 cm−1 , 1639 cm−1 , 2960 cm−1 (w), 3300 cm−1 . UV–vis: 269 nm ; 367 nm ; 664 nm .7.Uiso(H) = 1.2Ueq(C) for R2CH and R3CH H atoms and 1.5Ueq(C) for the methyl H atoms. The O-bound H atoms were located based on a difference-Fourier map and refined isotropically  or using riding model (H2A and H6) with O—H = 0.82 Å. Water H atoms were freely refined.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023002487/ex2068sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989023002487/ex2068Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023002487/ex2068sup3.tifFigure S1. FT-IR spectra of the complex. IR (KBr): 1026 cm-1(m), 1076 cm-1(w), 1134 cm-1(w), 1152 cm-1(m), 1197 cm-1(m), 1286 cm-1(w), 1317 cm-1(m), 1370 cm-1(m), 1450 cm-1, 1455 cm-1(m), 1535 cm-1(m), 1600 cm-1, 1639 cm-1, 2960 cm-1(w), 3300 cm-1. DOI: 2248369CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Hepatic fibrosis is a common consequence of chronic liver diseases without approved antifibrotic therapies. Long noncoding RNAs (lncRNAs) play an important role in various pathophysiological processes. However, the functions of certain lncRNAs involved in mediating the antifibrotic role remain largely unclear.4) or bile duct ligation (BDL) to investigate the function of lnc-Helf in liver fibrosis.The RNA level of lnc-High Expressed in Liver Fibrosis (Helf) was detected in both mouse and human fibrotic livers. Furthermore, lnc-Helf-silenced mice were treated with carbon tetrachloride  and activated hepatic stellate cells (HSCs). In vivo studies showed that silencing lnc-Helf by AAV8 vector alleviates CClOur results unveil a lnc-Helf/PTBP1/PIK3R5/AKT feedforward, amplifying signaling that exacerbates the process of hepatic inflammation and fibrosis, thus providing a possible therapeutic strategy for hepatic fibrosis.The online version contains supplementary material available at 10.1186/s11658-023-00492-3. Hepatic fibrosis is a common consequence of chronic liver diseases caused by various etiologies, such as chronic viral hepatitis B and C, cholestasis, or alcohol abuse , 2. If nFibrosis is characterized by the excessive deposition of extracellular matrix (ECM) components , 2. Acti4) or bile duct ligation (BDL)-induced proinflammatory M1 macrophage polarization and pyroptosis [The enormous transcripts transcribed from the mammalian genome are non-coding RNAs (ncRNAs) . Althougroptosis . Moreoveroptosis . In addiroptosis . DespiteIn this study, we identified a novel lnc-High Expressed in Liver Fibrosis (Helf) that is increased in mouse and human fibrotic livers and systemically investigated its regulatory and functional role in hepatic inflammation and fibrosis.Study population analysis was conducted as previously reported . BrieflyThree pathologists blinded to the study protocol scored liver fibrosis according to the meta-analysis of histological data in viral hepatitis (METAVIR) fibrosis staging system. The clinical characteristics were summarized in Additional file 4, 60 mice were randomly separated into six groups: AAV8-negative control (NC), NC + CCl4, AAV-lnc-Helf-shRNA-1#, AAV-lnc-Helf-shRNA-1# + CCl4, AAV-lnc-Helf-shRNA-2#, and AAV-lnc-Helf-shRNA-2# + CCl4 (n = 10). The adeno-associated virus was injected 2 weeks after the first injection of CCl4 via the tail vein (2 × 1011 pfu/mouse). NC + CCl4 group, lnc-Helf-sh1# + CCl4, and lnc-Helf-sh2# + CCl4 group mice were treated with CCl4 (0.2 ml/kg) twice per week for additional 6 weeks after the virus was injected. AAV8-NC, AAV-lnc-Helf-shRNA-1# and AAV-lnc-Helf-shRNA-2# group mice were injected with an equivalent amount of olive oil. For hepatic fibrosis model induced by BDL, 90 Balb/c mice were randomly separated into six groups: AAV8-NC, NC + BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + BDL, lnc-Helf-sh2#, and lnc-Helf-sh2# + BDL (n = 15). Mice were injected with AAV8-NC or AAV8-lnc-Helf-shRNAs virus (2 × 1011 pfu/mouse) via tail vein 2 days before sham operation or bile duct ligate operation. A total of 21 days after the operation, serum and liver specimens were collected for further analyses.All animal work was conducted according to the guidelines approved by the Animal Care and Use Committee of Tianjin Medical University. Eight-week-old Balb/c male mice were purchased from Beijing HFK bioscience . For hepatic fibrosis model induced by CCl4, AAV-lnc-Helf-shRNA-1# + CCl4, and AAV-lnc-Helf-shRNA-2# + CCl4 (n = 3) was qualified by Agilent 2100 bioanalyzer  and subsequently screened on the BGIseq500 platform . The threshold was fold change > 2 and padj < 0.05. The sequencing data have been deposited in NCBI GEO database: GSE182485 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE182485).RNA harvested from liver tissues of mice treated with NC + CClMouse primary HSCs were isolated from 40-week-old male Balb/c mice by in situ ethylene glycol tetra-acetic acid (EGTA)/Hank’s balanced salt solution (HBSS) solution, pronase, collagenase perfusion, further digestion, and Histodenz gradient centrifugation as previously reported , 19. Mor7 cells were lysed. RNA-bound beads were mixed to the lysates and incubated for 1 h at 4 °C. Followed by washing with wash buffer, we added elution buffer to the beads, mixed and incubated at 37 °C with agitation for 30 min. Supernatant was removed for downstream analysis .For biotinylated RNA pull-down assay, lnc-Helf was transcribed in vitro with T7 RNA polymerase . Then, T4 RNA ligase was used to attach a single biotinylated nucleotide to the 3′ terminus of an RNA strand at 16 °C for 16 h . The labeled RNA was bound to the Streptavidin Magnetic Beads at 25 °C incubating for 30 min. The mixture was washed with 20 mM Tris (pH 7.5) two times. Liver single cell suspensions of mice were prepared and total of 2 × 10RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit  according to the manufacturer’s instructions. Briefly, mouse liver single cell suspensions and RAW 264.7 cells were lysed by RIP lysis buffer and stored at −80 °C. A total of 5 µg of the antibody (anti-PTBP1 and control IgG) was added to magnetic beads and incubated with rotation for 30 min. Then, the RIP lysate was centrifuged at 14,000 rpm for 10 min at 4 °C. The beads were added to the lysates and RIP immunoprecipitation buffer and incubated at 4 °C for overnight. Next, RIP buffer was briefly centrifuged to remove the supernatant. The purified RNAs were used for cDNA synthesis and evaluated by quantitative reverse transcription polymerase chain reaction (qRT–PCR).t-test (two group comparison), and p < 0.05 indicated a significant difference.Data were expressed as mean ± standard error of the mean (SEM) with at least three independent experiments. All statistical analyses were conducted using SPSS 23.0 . Statistical analysis was conducted using either one-way analysis of variance (more than two groups) or Student’s Details on other materials and methods are provided in the Additional file 4 for various time periods, and the data revealed that the level of lnc-Helf was markedly increased with persistent injury  staining, Masson staining, Sirius red staining, and immunohistochemistry (IHC) for α-SMA, COL1α1, and TGFβ, and the data revealed that lnc-Helf silencing alleviated hepatic fibrosis induced by CCl4   and proliferation  in the livers  assay to identify the proteins binding with lnc-Helf Fig. B. Moreov4-treated lnc-Helf-shRNAs mice compared with CCl4-treated AAV8-NC mice  The fold change of lnc-Helf was shown according to the microarray data.  The nucleotide sequence of mouse lnc-Helf and human lnc-HELF. (D) The comparison between human and mouse lnc-Helf sequences. (E) The nuclei or cytoplasm of primary HSCs was isolated, and qRT–PCR analysis detected the expression of lnc-Helf, Neat1, and Gapdh. (F) The intracellular localization of lnc-Helf in primary HSCs was measured by RNA-FISH assays; scale bar is 10 μm. (G) qRT–PCR analysis of lnc-Helf in AML12 cells, LX-2 cells, RAW264.7 cells, BMMs, and HUVECs (H) qRT–PCR was used to assess the expression of lnc-Helf and Acta2 in primary HSCs cultured at day 2 treated with 10 ng/ml TGF-β for 24 h. (I–L) The correlations of lnc-HELF, ACTA2, COL1α1, ALT, and AST were assessed by Pearson correlation analysis, n = 34. *p < 0.05, unpaired Student’s t test (H). Fig. S2, related to Fig. S2. Mice were divided into six groups: AAV8-NC, NC + CCl4, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4, lnc-Helf-sh2#, and lnc-Helf-sh2# + CCl4. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 weeks after the first injection of CCl4 via tail vein. After CCl4 treatment for 8 weeks, (A–D) the volcano map and GO analysis of differentially expressed mRNAs in lnc-Helf-sh1# + CCl4 mice and lnc-Helf-sh2# + CCl4 mice are shown, compared with NC + CCl4 mice. Fig. S3, related to Fig. S2. Mice were divided into six groups: AAV8-NC, NC + CCl4, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4, lnc-Helf-sh2# and lnc-Helf-sh2# + CCl4. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 weeks after the first injection of CCl4 via tail vein. After CCl4 treatment for 8 weeks (A), IHC for COL1α1 and TGFβ are shown; scale bar is 100 μm for 40× and 400 μm for 10× magnifications . (B) qRT–PCR was used to assess the expression of Acta2, Col1α1, Mmp2, Timp1, and Tgfβ1.  Serum ALT and AST was examined. Data are presented as mean ± SEM.*/#p < 0.05. *p < 0.05 for AAV8-NC. #p < 0.05 for NC + CCl4, one-way ANOVA (B-D). Fig. S4, related to Fig. S2. Mice were divided into six groups: AAV8-NC, NC + CCl4, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4, lnc-Helf-sh2#, and lnc-Helf-sh2# + CCl4. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 weeks after the first injection of CCl4 via tail vein. After CCl4 treatment for 8 weeks, (A) western blot was used to determine the expression of CD11b, TNF-α, IL-1β, and MCP1. GAPDH was used as an internal control.  qRT–PCR was used to assess the RNA level of proinflammatory genes  and proliferation-related genes  in livers of each group. (D) IHC for CD11b, F4/80, TNF-α, IL-1β, LY6C, and PCNA; scale bar is 100 μm for 40× and 400 μm for 10× magnifications. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for AAV8-NC. #p < 0.05 for NC + CCl4, one-way ANOVA (B and C). Fig. S5, related to Fig. S3. Mice were divided into six groups: AAV8-NC, NC + BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + BDL, lnc-Helf-sh2# and lnc-Helf-sh2# + BDL. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 days before sham operation or bile duct ligate operation via tail vein. After 21 days of operation, (A) qRT–PCR was used to examine the RNA level of lnc-Helf in livers of each group.  Serum AST and ALT was examined. (D) qRT–PCR was used to assess the RNA level of Pcna, Ki67, Cyclin D1, and Bcl-2 in livers of each group. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for AAV8-NC. #p < 0.05 for NC + BDL, one-way ANOVA (A-D). Fig. S6, related to Fig. S4. (A) Quantitative analysis of western blot of Fig. S4B. (B) Primary HSCs at day 2 were infected with lenti-control and lnc-Helf-shRNA or lenti-lnc-Helf for 48 h, following the treatment with 10 ng/ml TGF-β1. The expression and location of COL1α1 was assessed by confocal microscopy. Scale bar is 20 μm. (C) Quantitative analysis of western blot of Fig. S4F. (D) Primary HSCs at day 2 were infected with lenti-control and lenti-lnc-Helf for 48 h, following the treatment with 10 ng/ml TGF-β1. The expression and location of COL1α1 was assessed by confocal microscopy. Scale bar is 20 μm. (E–G) LX-2 cells were infected with lenti-control or lenti-lnc-HELF for 48 h, following the treatment with TGF-β1 for 24 h. qRT–PCR was used to assess the expression of lnc-HELF, ACTA2, COL1α1, COL1α2, TIMP1, TGF-β1, PCNA, KI67, and CYCLIN D1 (E); western blot was used to determine the expression of the protein level of α-SMA, MMP2, CYCLIN D1, CYCLIN B1, and PCNA (F). GAPDH was used as an internal control. Cell proliferation was detected by CCK8 (G). (H) Primary HSCs at day 3 were infected with lenti-control and lenti-lnc-Helf for 96 h, cell proliferation was detected by CCK8. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for LV-control. #p < 0.05 for LV-control + TGF-β1, one-way ANOVA  and unpaired Student’s t test (G and H). Fig. S7, related to Fig. S5. (A) Mouse primary HMs transfected with siRNA for 24 h, qRT–PCR analysis was used to detect the RNA level of Mrc1, Arg1, Cd163, and Il10. (B) HMs were transfected with pcDNA3.1-lnc-Helf or pcDNA3.1 for 48 h, qRT–PCR was used to assess the expression of Mrc1, Arg1, Cd163, and Il10. (C) RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 48 h, qRT–PCR was used to assess the expression of lnc-Helf, Tnf-α, Mcp-1, Il-1β, Il-6, Pcna, Ki67, Cyclin D1, Cyclin E1, Mrc1, Arg1, and Il10. (D) RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 48 h following treatment with 20 ng/ml IFN-γ for 24 h. Mature supernatant IL-1β level was detected by ELISA. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for pcDNA3.1. #p < 0.05 for pcDNA3.1 + IFN-γ, unpaired Student’s t test (A–C) and one-way ANOVA (D). Fig. S8, related to Fig. S6. (A) Mice were divided into six groups: AAV8-NC, NC + BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + BDL, lnc-Helf-sh2#, and lnc-Helf-sh2# + BDL. Western blot was used to determine the protein level of phos-AKT (Thr308), phos-c-Raf1 (Ser259), phos-GSK-3β (Ser9), and AKT in liver tissues of each group. (B) LX-2 cells were infected with lenti-control or lenti-lnc-HELF for 48 h following treatment with 10 ng/ml TGF-β1 for 24 h. Western blot was used to determine the protein level of phos-AKT (Thr308) and AKT. GAPDH was used as an internal control. (C) RAW264.7 cells treated with or without the AKT inhibitor MK2206 were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 72 h. qRT–PCR was used to detect the expression of lnc-Helf, Tnf-α, Mcp-1, Il-1β, Il-6, Pcna, Cyclin D1, and Cyclin E1. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for pcDNA3.1 + DMSO. #p < 0.05 for pcDNA3.1-lnc-Helf + DMSO, one-way ANOVA (C). Fig. S9, related to Fig. S7. The expression and location of α-SMA in lnc-Helf-increased HSCs simultaneously transfected with siPTBP1 was assessed by confocal microscopy. Scale bar is 20 μm. Fig. S10, related to Fig. S8.  Prediction of the interaction probabilities between lnc-Helf and PTBP1 using the RPIseq and prediction of lncRNA–protein interactions database. (C) qRT–PCR was used to detect the RNA level of PTBP1 in livers of healthy people (n = 6) and fibrotic patients (n = 28).  The correlation of PTBP1, lnc-HELF, and PIK3R5 was assessed by Pearson correlation analysis, n = 34. (F–I) Mice were divided into six groups: AAV8-NC, NC + CCl4/BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4/BDL, lnc-Helf-sh2#, and lnc-Helf-sh2# + CCl4/BDL. qRT–PCR was used to detect the RNA level of Pik3r5 and Ptbp1 in livers of each group. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for AAV8-NC. #p < 0.05 for NC + CCl4/BDL, one-way ANOVA (F–I). Fig. S11, related to Fig. S8.  Primary HSCs at day 2 were infected with lenti-control or lnc-Helf-shRNA or lenti-lnc-Helf for 48 h, following the treatment with TGF-β1 for 24 h. qRT–PCR was used to detect the RNA level of Pik3r5. (C) LX-2 cells were infected with lenti-control or lenti-lnc-HELF for 48 h, following the treatment of TGF-β1 for 24 h. qRT–PCR was used to assess the expression of PIK3R5. (D) Mouse primary HMs were transfected with lnc-Helf siRNA for 24 h following treatment of 20 ng/ml IFN-γ for 24 h. qRT–PCR was used to assess the expression of Pik3r5.  Mouse primary HMs and RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 48 h, qRT–PCR was used to assess the expression of Pik3r5. (G) qRT–PCR was used to assess the expression of Pik3r5 in lnc-Helf-overexpressed RAW264.7 cells simultaneously transfected with siPTBP1. Data are presented as mean ± SEM. */#p < 0.05. *p < 0.05 for LV-control or si-NC or pcDNA3.1or pcDNA3.1 + si-NC. #p < 0.05 for LV-control + TGF-β1/IFN-γ or pcDNA3.1-lnc-Helf + si-NC, one-way ANOVA  and unpaired Student’s t test (E and F). Fig. S12. Schematic diagram shows the function and mechanism of lnc-Helf in the progression of hepatic inflammation and fibrosis. Upon liver injury, increased lnc-Helf binds with PTBP1 to promote its interaction with PIK3R5 mRNA, resulting in increased stability and activating the AKT pathway, thus promotes HSCs and HMs activation and proliferation, which augments hepatic inflammation and fibrosis. Table S1. Clinical characteristics of patients. Table S2. The results of mass spectrometry from proteins pulled down by the sense and antisense of lnc-Helf. Table S3. Cloning primers for lnc-Helf. Table S4 siRNA sequences. Table S5 qRT–PCR primers. Table S6 RACE primers for lnc-Helf.Additional file 2: The results of mass spectrometry from proteins pulled down by the sense and antisense of lnc-Helf.


The ionic title compound consists of a 2:1 ratio of the tetrabutylammonium cation (1+) and the tetrachloridomanganate(II) anion (2–). The structure reported contains two dichloromethane solvent molecules co-crystallized per anion. 16H36N)2[MnCl4]·2CH2Cl2, is an ionic organic–inorganic hybride compound consisting of a tetrabutylammonium cation and a tetrachloridomanganate(II) anion in a 2:1 stoichiometric ratio. The cation contains a central nitrogen atom bonded to four n-butyl groups in a tetrahedral arrangement, while the anion contains a central MnII atom tetrahedrally coordinated by four chlorido ligands. It co-crystallized with two equivalents of dichloromethane solvent, CH2Cl2, to give the following empirical formula: [(C4H9)4N]2[MnCl4]·(CH2Cl2)2. The crystal structure is mainly stabilized by Coulombic interactions.The title compound, (C In conducting our experiments, we inadvertently obtained the disolvated title compound and determined its crystal structure. After reviewing the literature, we realised that no crystallographic data had yet been reported on either the non-solvated or solvated forms of this substance. The only crystallographic data related to this system was the powder X-ray diffraction data for the non-solvated form at 900 K after it had already undergone thermal decomposition (Styczeń a) for the anion, and .2. (8 d) both for the cation and the solvent molecule. The root-mean-square deviations from ideal dT symmetry for the anion, S4 symmetry for the cation and Cv2 symmetry for the solvent molecule amount to 0.0123, 0.0501 and 0 Å, respectively, as calculated with PLATON 4N+, consists of a central nitrogen atom tetrahedrally surrounded by ordered butyl groups, with N—C bond lengths ranging from 1.505 (12) Å to 1.511 (11) Å and C—N—C bond angles in the range of 105.8 (5)–111.7 (11)°. The complex anion MnCl42– is consistent with the structure previously published for the tetramethylammonium salt -109.81 (12)°. The Mn—Cl bond lengths are all 2.364 (2) Å.The structural formula shows a ratio of 2:1 for the tetrabutylammonium cation and the tetrachloridomanganate(II) anion, combined with two solvent molecules of dichloromethane Fig. 1. The aboorni 1998, 2000 ▸.The crystal structure Fig. 2 is stabiet al., 20094·4H2O  was dissolved in warm absolute ethanol (10–15 ml). Separately, two equivalents of white (C4H9)4NCl·H2O  were also dissolved in warm absolute ethanol (10–15 ml). The two ethanol solutions were then mixed, and the solution turned a light-green color. The ethanol was removed under reduced pressure with heating to produce a pale-green solid. The solid was recrystallized from dichloromethane/ether to give pale-green crystals. After drying the crystals under reduced pressure at 311 K, they were massed . They were analyzed by IR and elemental analysis. IR (cm−1): 2962m, 2943m, 2875m, 1484s, 1468m, 1378m, 1151w, 1025w, 881m, 749m, 732m. Analysis calculated for (C16H36N)2MnCl4: C, 56.38; H, 10.65, N, 4.11. Found: C, 56.47; H, 11.47, N, 4.04. X-ray quality crystals were obtained from a mixture of dichloromethane/ether during a reaction involving the non-solvated form of the title compound as the starting material.A similar protocol was followed as previously reported in the literature (Styczeń A and H9B) were refined with half-occupancy.Crystal data, data collection and structure refinement details for the reported structure is summarized in Table 110.1107/S2414314623006107/wm4191sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623006107/wm4191Isup2.hklStructure factors: contains datablock(s) I. DOI: 2280618CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E)-2-[(benzo[d]thiazol-2-ylimino)methyl]-5-(diethylamino)phenol and its reaction product with triphenylborane are described. In compound 2N-BzEt, one of the ethyl groups and the benzothiazole ring are disordered over two sets of atomic sites with major occupancy components of 0.822 (5) and 0.843 (2), respectively.The molecular and crystal structure of ( E)-2-[(benzo[d]thiazol-2-ylimino)methyl]-5-(diethylamino)phenol  and its unexpected reaction product with triphenylborane, 2,2-diphenyl-1,3-dioxa-2-borata-1,2-dihydronaphthalene  are described. For 2N-BzEt, the hydroxyl group is involved in an intramolecular hydrogen bond with the imino nitrogen atom and the C=N bond displays an E configuration. The crystal packing is characterized by layers of inversion dimers parallel to the (10a-axis direction formed through C—H⋯O interactions. Complex (I) crystallizes with two molecules (A and B) in the asymmetric unit, which differ in the orientation of the ethyl groups. The 1,3-dioxa-2-borata-1,2,3,4-tetrahydronaphthalene ring displays a slight envelope conformation with the boron atom as the flap. In the crystal packing, chains of alternating A and B molecules formed by C—H⋯O hydrogen bonds run in the b-axis direction. The UV–vis absorption and emission properties of the compounds are discussed and their aggregation-induced emission properties are further investigated.The molecular and crystal structure of ( In the expected complex, boron would coordinate with the ligand through the oxygen atom of the hydroxyl group and the nitrogen atom of the imine group. But more surprisingly, the results of NMR and SC-XRD analysis indicated that the product obtained had structure (I) instead of the expected structure (II). This phenomenon can be explained by the fact that due to the simultaneous presence of Lewis acid BPh3 in the CHCl3 solvent, ligand 2N-BzEt is hydrolyzed and the boron atom is cyclized with the two oxygen atoms. To further elucidate this assumption, the interaction of 4-(diethylamino)-2-hydroxybenzaldehyde (2N-CHOEt) with BPh3 has been tested under similar experimental conditions. However, the TLC analysis results showed that no compounds were formed. The crystal structures and photophysical properties of the ligand 2N-BzEt and complex (I) are presented in this work.In this study, we intended to design a new boron(III) complex by replacing the amine component in the structure of boranils with 2-aminobenzothiazole to extend their π-conjugated system. From this idea,  Å for N3] and subtends a dihedral angle of 5.08 (7)° with phenyl ring C12–C17. The hydroxyl group O18—H18 is involved in an intramolecular hydrogen bond with the imino nitrogen atom N10 .Compound it Fig. 2. One of 10 Fig. 2. One of I) crystallizes in the triclinic space group PA (containing atom B1) and B (containing atom B2). In molecule A, the ethyl groups are on a different side of the ring to which the diethylamino group is attached, whereas in molecule B both ethyl groups are on the same side. The 1,3-dioxa-2-borata-1,2,3,4-tetrahydronaphthalene ring shows a slight envelope conformation with the boron atom as the flap. For molecule A, the deviation of atom B1 from the best plane through the ring is 0.315 (3) Å, for molecule B the deviation for B2 is 0.301 (3) Å. For molecule A, this boron-containing plane makes dihedral angles of 84.96 (14) and 81.09 (12)° with phenyl rings C8–C13 and C14–C19, respectively. For molecule B, the boron-containing plane makes angles of 77.83 (14) and 81.92 (12)° with phenyl rings C31–C36 and C37–C42, respectively.Complex  and thiazole (S1/C2/N3/C4/C9) rings [centroid–centroid distance = 3.7856 (13) Å]. The dimers form layers parallel to the , both molecules A and B are linked by a C24—H24⋯O2 hydrogen bond  the S—C—N=C torsion angle averages around ±180° (±ap or trans), while for 17 entries this torsion angle is close to 0° . For one entry  is caused by the incorporation of the terminal C—C bond of the search fragment into an indole ring. For 2N-BzEt this torsion angle is 177.55 (15)° for the major component of the benzothiazole ring and −2.6 (4)° for the minor component.A search of the Cambridge Structural Database ]. The average B—O(phenyl) and B—O distances are 1.498 (10) and 1.543 (30) Å, respectively [1.507 (3) Å and 1.560 (3) Å in (I)].A search for crystal structures containing a 1,3-dioxa-2-borata-1,2,3,4-tetrahydronaphthalene fragment in which the boron atom bears two additional carbon atoms resulted in five hits with refcodes ALUBOA  at 10 µM in chloroform solvent are shown in Fig. 82N-BzEt absorbs at 436 nm, while complex (I) shows absorption at 347 nm, which is a small shift from that of 2N-CHOEt (343 nm). The absorption peaks (343 nm and 347 nm) are attributed to the π–π* transition of the aromatic ring. Under a UV lamp with a 365 nm excitation wavelength, a solution of 2N-BzEt fluoresces green, while a solution of complex (I) shifts towards blue. In addition, this complex exhibits a longer emission wavelength and greater fluorescence intensity than that of 2N-CHOEt, demonstrating that complexation with boron can improve fluorescence properties compared to the free ligand.The UV–vis absorption and emission properties of the compounds 2N-BzEt and (I), we recorded the emission spectra of their 10 µM solutions in different fractions of water in a MeOH–water mixture. The results show that only compound 2N-BzEt is present as AIE active material  properties of ed Fig. 4, which a6.Synthesis of (E)-2-[(benzo[d2N-Bz).]thiazol-2-ylimino)methyl]-5-(diethylamino)phenol  in 10 mL of ethanol in a pressure tube was stirred at 348 K for 5 h. After cooling to room temperature (RT), the brown–yellow precipitated powder was filtered off, washed consecutively with a cold ethanol (1 × 5 mL), diethyl ether (1 × 5 mL) and then dried under vacuum at 323 K for 3 h. The yield was 87% . Single crystals suitable for X-ray diffraction and other analysis were obtained by slow evaporation within 8 h from a concentrated chloroform/ethanol (2:1 v/v) solution at RT. 1H NMR : δ 12.71 , 8.96 , 7.88 , 7.77 , 7.43 , 7.30 , 7.26 , 6.31 , 6.19 , 3.43 , 1.23 .A solution of 4-(diethylamino)-2-hydroxybenzaldehyde  and benzo[Reaction of (E)-2-[(benzo[d2N-Bz) with triphenylborane]thiazol-2-ylimino)methyl]-5-(diethylamino)phenol  and BPh3  in 3 mL of chloroform in a pressure tube was stirred at 333 K for 24 h. After cooling down, single crystals suitable for X-ray diffraction and other analysis were obtained by slow evaporation within 8 h from a reaction solution at RT. The yield was 55% . 1H NMR : δ 9,49 , 7,81 , 7.53 , 7.46 , 7.26 , 6.27 , 6.07 , 3.41 , 1.21 .In other solvents: The experiments in other solvents such as toluene, THF, ethanol were conducted under the same conditions as in chloroform. The course of reaction was monitored by TLC analysis. The results indicated that no new products were formed after 24 h of reaction.2N-CHO) with triphenylborane.Reaction of 4-(diethylamino)-2-hydroxybenzaldehyde (Et A solution of 4-(diethylamino)-2-hydroxybenzaldehyde  and BPh3  in 3 mL of chloroform in a pressure tube was stirred at 333 K for 24 h. The TLC analysis results indicated that there was no signal of the new product.7.2) and 0.96 Å (CH3). Non-hydrogen atoms were refined anisotropically and hydrogen atoms with isotropic temperature factors fixed at 1.2 times Ueq of the parent atoms (1.5 for methyl groups). For the O—H group in 2N-BzEt, the SHELXL command AFIX 148 was used in combination with U(H) = 1.2Ueq(O). One of the ethyl groups in 2N-BzEt is disordered over two sets of sites with refined occupancies of 0.822 (5) and 0.178 (5). Also the benzothiazole group is disordered over two positions by a rotation of 180° resulting in refined occupancies of 0.843 (2) and 0.157 (2) for atoms S1 and N3.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989023008514/pk2698sup1.cifCrystal structure: contains datablock(s) Et2N-Bz, I. DOI: 10.1107/S2056989023008514/pk2698Et2N-Bzsup2.hklStructure factors: contains datablock(s) Et2N-Bz. DOI: 10.1107/S2056989023008514/pk2698Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023008514/pk2698Et2N-Bzsup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023008514/pk2698sup5.pngFig. S1. DOI: 2297768, 2297767CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S)-butan-2-yl-N-(4-x-phenyl)thiocarbamates, x = NO2, OCH3, F, and Cl were synthesized from reacting aryl isothiocyanate with (S)-2-butanol to form new chiral crystals as the basis for future research into their non-linear physical and potentially biological properties.Enantiopure ( S)-butan-2-yl N-(4-nitrophenyl)thiocarbamate, C11H14N2O3S, (I), (S)-butan-2-yl N-(4-methoxyphenyl)thiocarbamate, C12H17NO2S, (II), (S)-butan-2-yl N-(4-fluorophenyl)thiocarbamate, C11H14FNOS, (III), and (S)-butan-2-yl N-(4-chlorophenyl)thiocarbamate, C11H14ClNOS, (IV), all at 100 K, have monoclinic (P21) symmetry with two independent molecules in the asymmetric unit. The Flack absolute structure parameters in all cases confirm the absence of inversion symmetry. The structures display N—H⋯S hydrogen bonds, resulting in R22(8) hydrogen-bonded ring synthons connecting the two independent molecules. Despite the ring synthon, the packing follows two distinct patterns, with (I) and (IV) ‘pancaking’ along the b-axis direction, while the other two ‘sandwich’ in layers perpendicular to the b axis. Crystal morphologies were determined theoretically via the BFDH  model and agree qualitatively with the experimentally indexed results. One of the butyl substituent of (II) exhibits structural disorder.The structures of ( Similar thiocarbamates have been investigated previously for their biological activities –(IV): −0.02 (3), −0.04 (4), 0.17 (13), and 0.022 (14), respectively].Isothiocyanates were selected because of the ease with which the –N=C=S functional group can be reacted with amines or alcohols to form thioureas or thiocarbamates, which in turn are well suited for simple crystal-growth studies. The –3.via the thioamide {⋯H—N—C=S}2 into –et al., 1999D—H⋯A angles appear to deviate slightly more from a ‘straight’ geometry in compounds (I)ortho-C—H of the phenyl groups . The interaction is strong enough to also cause the phenyl rings to become coplanar with the synthon plane. In (II)ortho-C—H distance to the sulfur atoms to be of lesser importance than in (I)In each structure shown in Fig. 1b-axis direction, while the other two ‘sandwich’ in layers perpendicular to the b-axis, see Fig. 2The packing follows two distinct patterns, with (I)Packing of (II) The 4-x-phenyl moiety is approximately parallel to the ac plane. The c plane in (II)bc plane in (III)S)-butan-2-yl moieties. Short distances between the phenyl plane and a symmetry-related OCH3 group are seen in (II)Packing of (I) The bc planes. Each dimer stacks entirely like ‘pancakes’ along the short b-axis, with a separate stack for the 21 axis-related dimers. The dimers are inclined to b so that the NO2 group of (I)2–phenyl plane distances are not the same for the independent phenyl moieties and are measured at 2.99 (2) and 3.169 (16) Å in (I)x-groups, . One oxygen atom of the NO2 in (I)2 > F > OCH3 > Cl -butan-2-yl equivalent structures to (I)et al., 2023P21/c, exhibit very similar packing to that of (IV)PRS)-butan-2-yl-4-CH3 phenylthiocarbamate crystal builds out the Via a GOOGLE search (March 2023), ‘phenylthiocarbamate’ is found 9,370 times. Most of the compounds incorporate a center of symmetry, which is often compatible with an et al., 2010The structures of this report are not found in the Cambridge Structural Database -butanol . Using a hot oil bath, the reaction was run at 381 K for 24 h. Compound (II)S)-butanol . While stirring, triethylamine  was added. After 5 minutes, the aryl isothiocyanate  was added dropwise. The reaction was allowed to continue for 24 h at 358 K. Subsequently, for all four compounds, the vials, after allowing to cool, were covered with filter paper and left in a vacuum oven at 343 K. The crude product was purified by flash column chromatography, and eluted with 1:4 ethyl acetate/hexane. Fractions were collected in 13 × 100 mm test tubes and were spotted for thin layer chromatography to locate the product. The fractions containing the product were rotovaped in a 25 ml round-bottom flask. The solid found in low yields was redissolved in a 1:4 methanol/ethanol solution and crystals grew via slow evaporation. (I)1H NMR : δ 9.2638 , 8.1926 , 7.5520 , 5.5528 , 1.7044 , 1.4038 , 0.9634 . (II)1H NMR 2CO): δ 9.7438 , 7.5813 , 6.9070 , 5.4819 , 3.7847 , 1.7022 , 1.2948 , 0.9184 . (III)1H NMR : δ 8.8978 , 7.2240 , 7.0147 , 5.0768 , 1.7280 , 1.3461 , 0.9316 . (IV)1H NMR : δ 8.7150 , 7.2918 , 7.2130 , 5.5199 , 1.7269 , 1.3640 , 0.9472 .All chemicals were obtained from Sigma Aldrich. Compounds (I)6.Uiso(H) = 1.2 (1.5 for methyl groups) times Ueq(C). The nitrogen protons were refined positionally, with Uiso(H) = 1.2Ueq(N). The two phenyl groups of the independent molecules of (III)S)-butan-2-yl moieties appeared threefold disordered, requiring restraint of the displacement parameters with a SIMU 0.01 command. One atom (C8) was constrained to the same displacement parameter for each fraction with EADP. The disordered geometries were linked through a SAME command to the geometry of the ordered moiety of the other molecule, and distances of O1 to C8, C8B and C8C were restrained to be similar (SADI), all with default esds. The occupancies of the three fractions were 0.444 (4), 0.354 (4), and 0.202 (4).Crystal data, data collection, and structure refinement details are summarized in Table 510.1107/S2056989023002591/zl5044sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, New_Global_Publ_Block. DOI: 10.1107/S2056989023002591/zl5044Isup5.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023002591/zl5044IIsup6.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989023002591/zl5044IIIsup7.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989023002591/zl5044IVsup8.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989023002591/zl5044Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023002591/zl5044IIsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023002591/zl5044IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023002591/zl5044IVsup9.cmlSupporting information file. DOI: 2249641, 2249640, 2249639, 2249638CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: