This has potential implications for understanding HCV induced immune activation as well as oncogenesis.Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our  Flaviviridae family which infects approximately 58 million people worldwide and can mediate severe hepatic injury and progressive liver fibrosis through stimulating persistent inflammation and oxidative stress (Hepatitis C Virus (HCV) is a member of the e stress –5. Approe stress –9. Severe stress , ethnicie stress , 12 and e stress –15. Conve stress , Interlee stress –19, sevee stress , 21.via downregulation of Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB) activation (in vitro (in vivo (in vitro stimulates endoplasmic reticulum (ER) stress and leads to the induction of the Unfolded Protein Response (UPR) and autophagy. These cryoprotective responses function to resolve ER stress by reducing protein synthesis and increasing the protein processing capacity of the infected cell  activation as a tool to measure the effects of these proteins on NF-κB signalling in 293T, TZM-bl and the more physiologically relevant Huh7 cell line. Using plasmids that express HIV-1 or luciferase under the control of the HIV-1 LTR, we investigated the ability of HCV Env glycoprotein to modulate cellular transcription pathways that may down-modulate immune signalling and promote the establishment of chronic HCV infection.2/37°C. Similarly, J-Lat 10.6 cells were maintained in RPMI-1640 supplemented with 10% heat inactivated FBS  and grown at 5% CO2/37°C.Cell lines 293T, Huh7 and TZM-bl were cultured in DMEM. All media was supplemented with 10% heat inactivated FBS  and 1% L-glutamine (Gibco) and cells grown at 5% CO4 per well). At 24 h post seeding cells were transfected with 150 ng of ATF3 shRNA plasmids or the scrambled shRNA or 12 ng of HCV E1E2, HCV SE2, HIV-1 JRFL (R5), HIV-1 LAI (X4), EBOV Env expressed plasmids using the polyethyleneimine (PEI) protocol. At 73 h post transfection cells were washed with PBS and 1:1 mixture of the cell suspension and 0.4% trypan blue solution was prepared. Cell viability was determined using automated cell counter. Similarly, viable cell counts for 293T and TZM-bl cells stably transfected with E1E2 were measured via 0.4% trypan blue solution and compared with the non-transfected cells.For measurement of cell viability following transfection with shRNA and viral envelope expression plasmids, 293T cells were seeded in a 12 well plate  was added to One Shot Top10 bacteria (ThermoFisher), mixed and incubated on ice for 30 min then incubated at 42°C for 30 s followed by 30 s on ice. 0.5 mL Super Optimal broth with Catabolite repression (SOC) media (ThermoFisher) was added and incubated for 1 h at 37°C with 180 rpm shaking before being plated on antibiotic selective agar plates overnight. Colonies were picked and grown in 5 mL Brain Heart Infuction (BHI)/ampicillin (100 µg/ml) culture  for miniprep isolation (Qiagen), which was used to check plasmids before larger maxipreps were performed. For maxipreps (Qiagen), 250 mL BHI/ampicillin cultures were used and grown overnight using the same conditions as stated above.2/37°C. For pseudo-typed virus particle production, the ΔEnv backbone pNL4-3-luc was used at 2,000 ng per condition as well as either 2000 ng of the HIV-1 Env expression plasmid, 285 ng of the Ebola Env plasmid or 200 ng of HCV Env E1E2 expression plasmids. In all cases, plasmids were added to 300 µl of Opti-MEM and this was then added to 300 µl of diluted PEI solution, mixed and incubated at rt for 20 min. Media was removed from 10 cm dishes and replaced with 1 ml Opti-MEM, and following incubation, transfection mix was added to cells dropwise and mixed and incubated under cell culture conditions for 6 h. Following this incubation, transfection media was replaced with DMEM.HIV-1 JRFL (R5), HIV-1 LAI (X4), EBOV GP (Ebola) and HCV E1E2 Env (H77 isolate genotype 1a) were transfected into 293T cells in 10 cm dishes using the PEI protocol. For all transfections, PEI was diluted to 0.08 µg/µl in OptiMEM (Gibco) and all cells were incubated in 5% COvia capsid-p24 standardised input. Target cells were seeded in 96 well plates (1.5x104 cells/well) 24 h prior to infection. Media was removed from the plate before 50 µL required media was added, followed by 100 µL of virus stock. After 6 h of incubation an additional 100 µL of appropriate cell media was added to each well and 48 h later cells were lysed and luciferase activity was quantified using BMG Labtech FLUOstar Omega with 100 µl luciferase substrate (Promega) injected per well.Infections were performed on TZM-bl, Huh7 and 293T cell lines. Viral stocks were quantified The p24 assay was performed using recombinant p24 standard, p24 coating antibody , secondary conjugate  from Aalto Bio Reagents Ltd and according to their protocol. Detection was performed using ELISA-Light Immunoassay System with CSP and Sapphire-I Substrate/Enhancer (ThermoFisher). All samples were diluted using 0.1% Empigen/TBS. Luminescence from ELISA plates was measured using a BMG Labtech FLUOstar Omega luminometer with a reading interval of 1 sec/well.4 cells/well). When confluent, media was removed and 50 µL of Opti-MEM added to each well. Two µL Opti-MEM added per well with the following plasmid quantities: 12, 6 or 1 ng E1E2 or sE2 plasmid , 6 ng LTR and 1 ng Tat. PEI was diluted to 0.14 µg/µl in 200 µL Opti-MEM and 2 µL of the diluted PEI was added to the plasmid mix. The plasmid/PEI mix was incubated at rt for 30 min before being added to cells and incubating for 6 h at 5% CO2/37°C after which Opti-MEM/plasmid mix was removed and 250 µL DMEM added. Plates were measured for luciferase activity 48 h after transfection as described above. The following genotypes of HCV E1E2 Env were used – 1a, 1b, 2b, 3, 4, 5 and 6  protein expression was determined in 293T cells transfected with ATF3 shRNA expression plasmids, HCV E1E2 Env and scrambled shRNA and pCDNA controls using western blot. Following transfection, cells were washed in ice cold dPBS and lysed with radioimmunoprecipitation assays (RIPA) buffer that included a 1% protease inhibitor cocktail (Thermo Scientific) and the protein concentration was subsequently quantified by the Bradford assay using the Protein Assay kit (Bio-Rad). 30 µg of cell lysates were mixed with 4x NuPage LDS Sample Buffer and 10x NuPage reducing agent, incubated at 72°C for 10 mins and loaded onto polyacrylamide gels , and electrophoresed at 120 V for 60 min.Separated proteins were transferred to iBlot™ 2 PVDF Mini Stacks membranes (ThermoFisher) employing the iBlot 2 Dry Blotting system. The membranes were then blocked using the iBind solution kit (ThermoFisher) and incubated with primary and secondary antibodies using the iBind device  for 3 h. Antibodies used in this study included rabbit anti-ATF3 , rabbit anti-β-actin  and horseradish peroxidase (HRP) conjugated secondary antibody . Protein bands were visualized using Pierce™ ECL Plus Western Blotting Substrate (ThermoFisher).4 cells/well). At 24 h post seeding cells were transfected with either an E1E2 or sE2 expression plasmid containing a V5 tagged genotype 1A E1E2 or sE2 gene (expressing neomycin resistance) using the PEI protocol. Briefly, the transfection mix was prepared by diluting PEI to 0.08 µg/µl in 50 µL Opti-MEM to which 50 ng of envelope plasmid was added. This mix was incubated for 30 min at rt before being added to the well and incubated for 6 h in cell culture conditions, after which Opti-MEM/plasmid mix was removed and 1 mL DMEM was added. At 48 h post transfection media was refreshed using antibiotic selection (optimised as 400 µg/ml G418). Resistant cells were expanded before being tested for plasmid expression via FACS analysis (V5 directed Ab) and monitored periodically. TZM-bl-E1E2 and TZM-bl-sE2 cell lines were generated (E1E2: UKN1A20.8 EU155192. sE2: soluble E2_661 1A20.8.4). E1E2 and sE2 expression was confirmed via FACS staining of cells. In short, 2.5x105 cells were harvested 48 hours post transfection and were resuspended in 100 µl 4% paraformaldehyde solution for 20 minutes at 4°C. Cells were then washed twice in staining buffer (0.2 µm pore filtered PBS/1% heat inactivated FBS) and resuspended in fixation buffer (PBS/1% heat inactivated FBS/0.5% saponin) for 15 mins at room temperature. Cells were then resuspended in 50 µl fixation buffer containing the primary antibody (AP33 primary antibody at a 1:10 dilution) for 30 minutes at 4°C in the dark. Cells were washed with fixation buffer twice before being resuspended in 20 µl fixation buffer containing the secondary antibody (anti-mouse goat PE conjugate - 1:5 dilution), for 30 mins at 4°C in the dark. Cells were then washed and resuspended in 200 µl staining buffer at 4°C in the dark until FACS analysis. The AP33 antibody recognises E2 epitopes between aa412-423.TZM-bl cells were seeded in a 12 well plate  was then added for 5 mins before being removed and 0.6 mL lysis buffer (10 mL HB 2x supplemented with 400 µL 10% NP-40 and 9.6 mL H20) added for a further 5 min incubation. The cells were transferred to an Eppendorf microcentrifuge tube and centrifuged at 13,000 rpm for 60 s. The supernatant was removed and the pellet resuspended in 50 µL resuspension buffer . 60 µL salt buffer  was then added before the samples were incubated for 1 hr on a rotating wheel. 1 mL each resuspension buffer and salt buffer, supplemented with 40 µL PIC 25x  and 100 µL Phospho Stop 10x  added prior to use. The samples were centrifuged at 13,000 rpm for 10 min before protein concentration quantified via the Bradford assay. Once quantified, the samples were aliquoted and stored at -80°C. All steps of this protocol were undertaken either on ice or at 4°C. Bradford quantification was performed using Quick Start Bradford Reagent (Bio-Rad).TZM-bl and TZM-bl-E1E2 cells were grown to required confluency prior to extraction. Cells were washed with 10 mL cold PBS, followed by 10 mL cold PBS supplemented with 5 mM NaF and 1 mM NaThe DNA capture probe production and affinity purification were performed as described . BrieflyPeptides were analysed using nano-LC-ESI-MS/MS maXis Impact UHR-TOF  coupled with a UPLC Dionex UltiMate 3000 (ThermoFisher). Digests were separated by reverse-phase liquid chromatography using a 75 µm X 250 mm reverse phase column  in an Ultimate 3000 liquid chromatography system. Mobile phase A was 95% of 0.1% formic acid in water and 5% acetonitrile. Mobile phase B was 0.1% formic acid in acetonitrile. The runtime was 120 min. The digest (15 µL) was injected, and the organic content of the mobile phase was increased linearly from 4% B to 35% in 90 min and from 35% B to 90% B in 5 min. The column effluent was connected to a Captive Spray (Bruker). In survey scan, MS spectra were acquired for 0.5 s in the m/z range between 50 and 2200. The 10 most intense peptides ions 2+ or 3+ were sequenced. The collision-induced dissociation (CID) energy was automatically set according to mass to charge (m/z) ratio and charge state of the precursor ion. The MaXis and the Ultimate systems were piloted by Compass HyStar 3.2 (Bruker).Peak lists were created using DataAnalysis 4.1 (Bruker) and saved as MGF file for use with ProteinScape 3.1 (Bruker) with Mascot 2.4 as search engine (Matrix Science). Enzyme specificity was set to trypsin, and the maximum number of missed cleavages per peptide was set at one. Carbamidomethylation was allowed as fixed modification, oxidation of methionine, Gln–pyro-Glu and Carbamidomethylation (N-term) were allowed as variable modification. Mass tolerance for monoisotopic peptide window was 7 ppm and MS/MS tolerance window was set to 0.05Da. The peak lists were searched against a subset from the taxonomy homo sapiens of the UNIPROT database (162267 entries). Scaffold software  was used to validate MS/MS based peptide and protein identification. For each sample, two analyses were made and results coming from Mascot were merged before performing a second search with X!Tandem . Peptide identifications were accepted if above 75% probability to achieve an FDR less than 1% by the Scaffold local FDR algorithm. Protein identifications were accepted if above 11% probability to achieve an FDR less than 2% and contained at least 2 identified peptides. Proteins sharing significance peptide evidence were grouped into clusters. Spectral counting quantitative analysis was used to compare the samples. T-test with a significance level of p<0.05 and a normalization based on total spectra were applied to the results.5 cells per well). The media was replaced with 500 µL of Opti-MEM per well. 250 ng DNA was added to 300 µL Opti-MEM media and mixed with 300 µL 0.08 µg/µl PEI and incubated for 30 mins before added to the cells and incubated for 48 h. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). Reverse transcription of total RNA was performed using the Superscript III cDNA kit (ThermoFisher). Quantitative RT-PCR measuring relative expression of NF-κB , RELA , IFI16  and RBMX  was performed using SYBR green master mix, 1 µL primers, 4 µL nuclease free water and 5 µL of cDNA in total reaction volume of 20 µL. GAPDH  was used as a control housekeeping gene. Samples were amplified and detected using a GeneRoter (Qiagen). Reactions were prepared in triplicate for each sample. PCR reaction conditions: 50°C for 2 min, 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 10 s and a total of 40 amplification cycles were performed with the final extension being 60°C for 5 min. The results were normalised to GAPDH expression.293T cells were plated o/n in six well plates  supplemented with β-mercaptoehtanol. RNA was purified from lysates using RNeasy Plus Minikit (Qiagen), according to manufacturer’s instructions and eluted in 35 µl nuclease free water. RNA quality and purity were assessed using a Nanodrop spectrophotometer and Agilent Bioanalyzer, which indicated that all samples had an RNA Integrity Number (RIN) >9.3 (min 9.4 and max 10.0). RNA was quantified using Qubit high sensitivity RNA fluorometer. PolyA+ mRNA was purified from 35 µg total RNA using Dynabeads mRNA purification kit (ThermoFisher) and eluted in 15 µl of nuclease free water. Libraries were prepared using 30 ng polyA+ mRNA according to the Oxford Nanopore SQK-PCS-109 protocol in conjunction with EXP-PBC-001 barcoding kit to allow multiplexing of flow cells. Briefly, reverse transcription was performed using Maxima H Minus Reverse Transcriptase  and 5 µl of reverse transcription product was separated into 2x 50 µl reactions with Oxford Nanopore barcoded primers for amplification . Amplification products were pooled and purified using Ampure XP magnetic beads (Beckman Coulter) with a 0.45x ratio of beads to DNA volume. Three adapted libraries were sequenced per flow cell, using 100 ng of each library. Libraries derived from control pCDNA transfected cells and libraries derived from HCV E1E2 transfected cells were sequenced on the same flow cells to limit flow cell associated bias. Sequencing was performed using MIN-106 flow cells (R9.4.1 or R9.4 pores) on the Oxford Nanopore MinION sequencing platform. Run durations ranged from 12-24 h.293T cells were seeded at 4.8x10https://github.com/rrwick/Porechop). To confirm expression of HCV E1E2 in transfected cells, libraries were mapped to a database of viral genomes using Kraken2  and reads with a q-score <7 were discarded during basecalling. Each sequencing library was demultiplexed and adapter sequences were trimmed using Porechop  24 h prior to transfection. On day of transfection, medium was replaced with 100 µl Opti-MEM. Transfection mix was prepared by diluting PEI to 0.08 µg/µl in 50 µl Opti-MEM to which 300 ng of ATF3 shRNA plasmids or the scrambled shRNA or pCDNA control was added. The mix was incubated at rt for 30 min before adding to cells and incubated for 6 h after which the Opti-MEM/plasmid mix was removed and 1 ml DMEM added. After 48 h cells were re-transfected with 50 ng of LTR-Luc and 5 ng Tat expression plasmids, using the same conditions as described above. The plate was measured for luciferase activity 48 h after transfection. Similar was performed utilising a 96 well plate format (1.5x104 cells per well) following the same procedures with reagent volumes and quantities adjusted accordingly.Three ATF3 directed shRNA and a scrambled expression plasmid (pSUPER) were a kind gift from Dr Martin Janz  . 293T ceStatistical analysis was performed using GraphPad Prism 8 software unless specified otherwise. For RNAseq, statistical analysis was performed using R packages Voom/Limma. Data were analysed using the Kruskal-Wallis one-way analysis of variance followed by using Dunn’s analysis to perform paired multiple comparisons. Ns – non-statistical. * P<0.05. ** P<0.01.env) in conjunction with a plasmid expressing viral Env proteins was performed. When producing pseudo-typed lentiviral particles expressing the HCV envelope E1E2 a virus stock was generated that infects liver derived Huh7 cells but not TZM-bl cells, indicating HCV E1E2 protein expression . Transfection of 293T producer cells with a plasmid expressing the HIV-1 viral backbone minus the Env gene (Δ3 TCID50/ml input) and sampling viral p24 production  protein under drug selection . Plasmids were generated which expressed HCV E1E2 or sE2 carrying a V5 tagged epitope which allowed for testing transfected cell lines and confirming protein expression . This cell carries a single integrated HIV-1 genome that can be activated with Tumour Necrosis Factor α (TNFα). An array of plasmid constructs  were transfected into J-Lat-10.6 cells, which were subsequently activated with TNFα and monitored for viral p24 production    against three selected HIV-1 LTR promoter subtypes . Two concentrations of E1E2 or sE2 plasmids (12ng and 1ng) were co-transfected along with the LTR plasmids in conjunction with the Tat expressing plasmid. Transfection of LTR subtype A with E1E2 demonstrates the ability of E1E2 to down-modulate LTR activation  mutant plasmid was generated and used to transfect 293T cells. It was observed that LTR activity was down-modulated in the presence of E1E2 protein (ATG+) (P<0.05) but not when E1E2 protein expression was knocked-out (ATG – KO Mut) (-18) and “Transcription Regulation” , based on normalized spectral count analysis of the data, a large number of proteins were found to differentiate in binding to the HIV-1 LTR, with 22 proteins binding in the presence of HCV E1E2 and 34 proteins enriched in the control experimental conditions  strategy utilises a double stranded LTR DNA capture probe to pull-down proteins from cells transfected with pCDNA control plasmid or with plasmid expressing HCV E1E2  and analysed the nuclear extract from these cells. The LTR probe was generated as a PCR template of WT HIV-1 5’LTR   to identify whether the protein binding alterations observed correlated to alterations at the mRNA expression levels. Quantification assays were developed for each gene, but no differences were found in mRNA expression for NF-κB, RelA, IFI16 or RBMX   patterns between cells expressing the HCV E1E2 Env protein in comparison to cells transfected with the control vector, pCDNA (GEO accession: GSE163239). We selected to utilise the 293T cell line for differential gene expression analysis to provide consistency with results showing the effect of E1E2 expression on down-modulating HIV-1 LTR activity (section 3.2). Since the down-modulation of HIV-1 LTR activity in the presence of HCV E1E2 Env protein is similar between 293T and Huh7 cells we are confident our results are reflective of cell types infected with HCV. Initially, to demonstrate expression of E1E2, all the expression libraries were mapped to an HCV reference genome, highlighting expression of HCV derived genes only in the E1E2 transfected cells (is (PCA) –52  member 5 (HSPA5) gene encodes the binding immunoglobulin protein (BiP) and is the most significantly upregulated gene in our dataset 8A. BiP Based on the above finding that ATF3 (a major component to the ER stress pathway) was significantly up-regulated in the presence of E1E2 we aimed to identify whether knocking-down the expression of this specific gene could modulate the E1E2 inhibitory effect observed. This is also linked with previous reports that ATF3 negatively regulates NF-κB activity , 67, To To identify whether knocking-down ATF3 could modulate NF-κB dependent responses we transfected the above generated ATF3 knock-down cells with HIV-1 LTR-Luc (NF-κB dependent) and Tat expression plasmids and measured Luc activity 48 h later. Pre-transfection with the three ATF3 directed shRNA plasmids provided a higher level of Luc activity than with control plasmid alone (P<0.05)  can modulate NF-κB signalling, potentially leading to pronounced effects on an array of biological phenomena.We have demonstrated an increased effect by sE2 alone to down-modulate HIV-1 LTR activity. Soluble E2 (sE2) has been used previously as a means to map antibody epitopes , CD81-reHCV E2 has previously been documented to be capable of modulating several intracellular signalling pathways, including the MAPK/ERK , PI3/AKTThree members of the NF-κB family were detected exclusively [RelA and NF-κB2 ] or more abundantly [NF-κB1 ] in the HIV-1-LTR pull-down performed in the absence of E1E2. This suggests that, although the expression . Alternavia transforming growth factor β1 (TGF-β1) induction , PERK (double-stranded RNA-dependent protein kinase (PKR)-like ER kinase) and ATF6 (activating transcription factor 6) , 35. Thevia direct or indirect interaction with UPR effector proteins. One potential regulator is ATF3, a transcription factor that is involved in the host response to inflammation, infection and cancer , it is proposed that E2 may be the means by which HCV modulates NF-ĸB signalling (as part of the E1E2 heterodimer complex) and future via chromatin remodelling thus abolishing the histone deacetylase and methyltransferase related regulation  where HCV replication occurs in adjacent hepatocytes. Indeed in vitro culture systems have indicated that liver fibrosis induction can be enhanced in HIV-1 infected individuals through complex interactions of cell-types infected with either HIV-1 or HCV  . AdditioOverall, it is shown that HCV E2 possesses a wide-ranging capability to influence intracellular signalling events. The research presented here also demonstrates a novel consequence of HCV/HIV-1 co-infection in which E2 down-modulates LTR activity. Given that co-infection of the same cell is rare the major consequences to these findings will lie in the effect of the HCV E2 Env protein in majorly down-modulating NF-κB mediated pro-inflammatory as well as oncogenesis pathways in HCV infected individuals.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163239). All reagents generated will be available from the Lead Contact with a completed Materials Transfer Agreement.Data has been deposited in NCBI’s Gene Expression Omnibus and accessible through GEO Series accession number GSE163239 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein, we report a continuous flow process for the synthesis of 2,6-diisopropylphenol—also known as Propofol—a short-acting intravenous anesthesia, widely used in intensive care medicine to provide sedation and hypnosis. The synthesis is based on a two-step procedure: a double Friedel–Crafts alkylation followed by a decarboxylation step, both under continuous flow. Propofol  is a potent intravenous hypnotic agent, which is widely used for the induction and maintenance of anesthesia and for sedation in the intensive care units . PropofoImportantly, within the context of the COVID-19 pandemic, Propofol is extensively used to avoid cardio-pulmonary injuries for patients, who are mechanically ventilated by minimizing resistance to this mechanical ventilation . At the Although, most of the drugs are produced under batch conditions, continuous flow chemistry has emerged as a possible alternative for the production of active pharmaceutical ingredients (API) ,5,6, esp1) as the starting material to hamper the undesired alkylation at the para position. ..1) as th1), as starting material, and studied the Friedel–Crafts alkylation with IPA to introduce the two isopropyl substituents. At the outset of our study, we faced a solubility issue with the solvent mixture initially envisioned to introduce the substrate 1 (H2SO4/IPA/water) by a single inlet in a heated coil reactor. Hence, to avoid any precipitation in the tube reactor, which would jeopardize the development of the continuous flow synthesis of the target, we implemented a set-up composed of two inlets feeding a PFA coil reactor (ID = 1.6 mm) equipped with a T-shaped mixer: inlet A contained the substrate 1 in a homogeneous H2SO4/H2O mixture  in a H2SO4/H2O mixture as well (9:1). Importantly, the stock solutions were preheated at 35 °C to avoid any possible clogging before the injection within the reactor.Inspired by this contribution toward the batch synthesis of Propofol, we started our investigations on a continuous flow process from 4-hydroxybenzoic acid , as startR = 40 min and adjusting the reactor length accordingly (2 with very good isolated yields (entries 1 to 4). An optimum total flow rate of QT = 2.5 mL·min−1  provided 2 with 84% yield. Importantly, the reaction was scaled up to 200 mmol , without loss of efficiency and the pure 2 was delivered with a high yield (84%) after a short filtration over a pad of silica gel (entry 4) [Then, the influence of the flow rate—and hence the mixing efficiency—was studied by fixing the residence time at ordingly . As descentry 4) .1 into 2, we turned our attention to the final decarboxylation step to access Propofol. This second step, initially performed in batch with a solution of NaOH, 2.3 M in 2-ethoxyethanol at 130 °C for 12 h  for a better thermal transfer and a putative assistance in the decarboxylation event . The system was equipped with a back pressure regulator (BPR) set at 9.5 bars to reach high temperatures. Since flow systems offer the possibility to telescope reactions, our studies began with solvent mixtures including toluene, the solvent used to extract 2 from the final the previous step (vide supra). Among bases assessed, TMEDA proved to be efficient, leading to fairly decent NMR yields (entries 1 and 2) compared to DIPEA (entries 3 and 4): ca. 60% of expected Propofol product vs. >20%. Note that similar results to those of TMEDA were obtained with the inexpensive TEA (entries 5–7) and an optimum of 2 equivalents was required. In addition, reactions performed with TEA afforded cleaner reaction mixtures . Hence, the solvent optimization was pursued with TEA: switching from toluene/2-ethoxyethanol mixture to DMF (entry 9) or pure 2-ethoxyethanol (entry 10) allowed the formation of Propofol in very high yield (86%). Further optimization by increasing the temperature to 200 °C using 2-butoxyethanol allowed to reach 69% yield within 30 min (Q = 334 µL·min−1) at this higher temperature (entry 11). Thus, whereas the yield of the reaction is not formally improved, this parameter shall not be considered on its own under flow conditions. The reaction time is also an important factor that determines the productivity. Therefore, a residence time of 0.5 h at 200 °C appears more appealing than 3 h at 150 °C (entry 10 vs. entry 11).To achieve the synthesis of Propofol, the flow system was composed of a single inlet, containing a premixed solution of The impurities profile for pharmaceutical compounds is fundamental for safety reasons, especially for intravenous drugs such as Propofol. Industrials processes allowing the Propofol have revealed the presence of numerous side products—which were detected by gas chromatography —resultinIn conclusion, we have reported herein the first continuous flow synthesis of the strategic Propofol anesthetic, recognized by the WHO within its essential list of medicines. This easy, practical, and safe two-step procedures afforded the Propofol in 70 min (vs. 16 h in batch) with an overall yield of 58% (84% yield for the Friedel–Crafts step and 69% yield for the decarboxylation step) close to the 64% described in batch (85% yield for the Friedel–Crafts step and 75% yield for the decarboxylation step). The final product is obtained with an excellent purity as shown by GC analysis. In addition, this practical continuous flow synthesis minimizes the exposure of the practitioner to concentrated sulfuric acid thanks to the use of a continuous flow reactor and a smoother quench of the acidic solutions (less exothermic). Finally, the decarboxylation reaction was smoothly addressed within 30 min thanks to an efficient and easy heat transfer. We do believe that this synthesis will pave the way for industrial endeavours to push this synthesis from a laboratory scale continuous flow synthesis to a highly productive continuous flow production manifold.1H NMR spectra and 13C NMR spectra were recorded on a Bruker Advance III 300  at 300 MHz and 76 MHz, respectively , multiplicity , coupling constant (Hz), and integration. Chemical shifts are reported in ppm relative to the signals corresponding to residual non-deuterated (CDCl3: δ = 7.26 ppm). Analyses were performed on GC-FID Thermoscientific trace 1310 equipped with Durabond DB-5MS , the He vector gas flow rate of 1 mL·min−1 in split mode (50 mL·min−1) and the inlet injector temperature of 250 °C. As FID detector, the air flow rate is at 350 mL·min−1 with vector gas at 35 mL·min−1. Samples analyses were done after 2 min at 50 °C, at a temperature rate of 10 °C·min−1 for 20 min reaching 250 °C. All GC-sample were prepared using grade HPLC solvents from Fisher . Distillation were performed on a Büchi Kugelrohr. The flow reactor system is specified for each cases between the AFR™ Corning® module pump device, the Vapourtec® R4 heated system and R2S pump system.All materials were purchased from commercial suppliers. Unless specified otherwise, all reagents and solvents were used as supplied. 1)  in H2SO4/H2O  and a stock solution of isopropyl alcohol  in H2SO4/H2O  were heated at 35 °C and pumped using the AFR™ Corning® module pump at a flow rate of 1.25 mL·min−1 for each solution through a T-mixer heated at 60 °C and a 100 mL coil PFA reactor also heated at 60 °C. The output of the reactor was collected into a stirred solution of H2O/toluene (1:1) at room temperature. The setup was eluted by a solution of H2SO4/H2O (9:1). The layers were separated and the aqueous phase was extracted with toluene. The combined organics phases were washed with brine, dried with MgSO4, and concentrated under vacuum. Flash chromatography afforded the desired product 2 as a white solid .A stock solution of 4-hydroxybenzoic acid  δ 7.87 , 3.17 , 1.31 . 13C NMR  δ 172.64, 155.15, 133.73, 126.50, 121.47, 27.22, 22.60. Melting point: 139–142 °C. GC retention time: 19.02 min.2  in 2-butoxyethanol (500 µL) was added triethylamine . The mixture was stirred at room temperature and pumped by R2S Vapourtec® pump device at a flow rate of 334 µL·min−1 through a 10 mL copper coil reactor at 200 °C and through a 1 mL copper coil reactor at room temperature. The setup was eluted by 2-butoxyethanol. The output of the reactor was collected into a stirred solution of brine/cyclohexane [4 and concentrated under vacuum to afford the crude Propofol with an excellent purity. A subsequent bulb-to-bulb distillation of this residue  is possible to further purify the Propofol, which is obtained as a colorless liquid (58% yield).To a stock solution of 3,5-diisopropyl-4-hydroxybenzoic acid lohexane . The org1H NMR  δ 7.07 , 6.96–6.85 , 4.78 , 3.17 , 1.28 . 13C NMR  δ 149.97, 133.66, 123.48, 120.66, 27.18, 22.78. GC retention time: 13.70 min.


GD562 exhibited improved inhibition of the cellular N-terminal methylation levels of both the regulator of chromosome condensation 1 and the oncoprotein SET with an IC50 value of ~50 µM in human colorectal cancer HCT116 cells. Notably, the inhibitory activity of GD562 for the SET protein increased over 6-fold compared with the previously reported cell-potent inhibitor DC541. Furthermore, GD562 also exhibited over 100-fold selectivity for NTMT1 against several other methyltransferases. Thus, this study provides a valuable probe to investigate the biological functions of NTMT1.Protein N-terminal methyltransferase 1 (NTMT1) recognizes a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) and transfers 1–3 methyl groups to the N-terminal region of its substrates. Guided by the co-crystal structures of NTMT1 in complex with the previously reported peptidomimetic inhibitor DC113, we designed and synthesized a series of new peptidomimetic inhibitors. Through a focused optimization of DC113, we discovered a new cell-potent peptidomimetic inhibitor GD562 (IC S-adenosyl-L-methionine (SAM). Protein N-terminal methyltransferase 1/2  are the two reported members of this family that recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) . Hoeability  confirmeCompared to DC113, DC541 exhibited about 10-fold enhanced cellular inhibition with a ~3-fold decrease in its inhibitory activity on recombinant NTMT1 , suggest2 was synthesized through the standard amide coupling of HCl•Lys(Boc)-COMe with Naphthalene-Pro. Subsequent hydrolysis of 2 provided 3. The final peptidomimetic inhibitors 1a–k were prepared by the reactions of various amines with 3, followed by the acidic treatment with 4 N HCl in 1,4-Dioxane.All the peptidomimetic inhibitors were synthesized in the solution phase, as shown in 2O, MeOH/H2O, 0 °C to r.t.; (c) R-NH2, HBTU, HOBt, DIPEA, DMF, r.t.; (d) 4N HCl in dioxane, 0 °C to r.t.Reagent and conditions: (a) HCl•Lys(Boc)-COMe, HBTU, HOBt, DIPEA, DMF, r.t.; (b) LiOH•H1a and 1b with higher cLogP values than DC541  compared with 1c. As N-methylation has been proven to improve the cell permeability of peptides [1d to produce 1i, which resulted in a 2-fold decreased inhibition. To explore the possible interaction with Tyr215 [1j (IC50 = 2.5 ± 0.09 µM), resulting in a ~3-fold inhibition reduction compared to 1d. Then, we switched the substitution position of the phenyl ring from the para to the meta position to produce 1k (IC50 = 0.69 ± 0.08 µM), rescuing the inhibition with the comparable activity as the parent compound 1d.Inhibitory activities of all synthesized compounds were determined with an established SAHH-coupled fluorescence assay ,15. To ian DC541 . Both copeptides , we intrh Tyr215 , we synt50 values correlated well with the docking scores yielding an R2 of ~0.7 (1j (GD590) docked with a similar binding mode as DC541 and DC113. For example, the ε-NH2 group of the Lys3 side chain in 1a (GD556) formed electrostatic interactions with Asp177 and Asp180, while the backbone carbonyl group interacted with Tyr215 through a hydrogen bond  facilitated interactions with Asp 180, Asp 177, and Tyr215, with an additional π-π interaction between the fourth position phenyl ring and Tyr215  ,20. The  of ~0.7 A. All pegen bond B. In conto DC541 C. In supd Tyr215 D.1a–h) [Gbind) was again observed with a Pearson R of 0.71  distances between stacked benzene rings are often ~5 Å apart [Gbind and increasing COM distances between substituent rings and Tyr215, measured from the MSλD trajectories. Surprisingly, as the carbon linker was extended, the consistent orientation of the methylene hydrogens pointed towards the π surface of Tyr215  for a subset of compounds (1a–h) ,22. A go of 0.71 . Experimractions . In mole Å apart . Figure f Tyr215 . Though 50 < 2 μM) in HCT116 cells through alamaBlue assay, as HCT116 cells are known for their high expression of NTMT1 [1a, 1b, 1g, and 1h) and 1k displayed cytotoxicity, with GI50 values ranging from 48 μM to 135 μM  exhibited improved cellular inhibition on me3-RCC1 with an IC50 of ~50 µM  [−6 cm/s, comparable to the literature value (8.8 ± 0.53 × 10−6 cm/s) [1c–e with variable carbon length showed the lowest permeability values ranging from 0.17 × 10−6 cm/s to 0.26 × 10−6 cm/s, which was about two-fold less than the cyclohexyl analog 1f with a permeability value of 0.47 × 10−6 cm/s. This suggests that acyclic rings have better cell permeability than aromatic rings. Methylation of the C-terminal amide of 1d to 1i resulted in a higher cLogP, which was also evident in a roughly two-fold improvement in cell permeability. As expected, the biphenyl analogs 1j and 1k had the higher cell permeability values of 0.9 × 10−6 cm/s and 1.3 × 10−6 cm/s due to the improved lipophilicity of the additional phenyl group. This trend of the permeability values from the PAMPA assay is proportional to that of the cLogP values, with 1j and 1k displaying the highest cLogP values of 3.8. To understand the discrepancy between biochemical IC (PAMPA) ,29,30. TSince GD562 exhibited the highest cellular inhibition activity and lower cytotoxicity among all tested peptidomimetic inhibitors , the com50 as me3-RCC1. Besides, GD562 exhibited improved selectivity over DC541 regarding PRMT1. Noticeably, the cellular inhibition activity of NTMT1 by GD562 is not optimal since there is still a ~50-fold difference between its enzymatic and cellular IC50 values. Based on the PAMPA result, permeability alone did not explain the discrepancy between the biochemical and cellular IC50 values. Future directions include further improving the cellular potency of these compounds by adding substituent groups to the C-terminal phenyl ring, or testing new heterocycles at the C-region. Meanwhile, fluorescent labeling GD562 to monitor its cellular distribution would also be informative, as NTMT1 is predominantly located in the nucleus [50 value of ~50 µM. Therefore, it can serve as a valuable tool to investigate the biological functions of NTMT1 in cells and as a new lead compound for further optimization. In summary, a series of new peptidomimetic inhibitors were designed, synthesized, and evaluated based on the structures of previously reported inhibitors. We successfully optimized the cellular inhibition activity of the peptidomimetic inhibitor by introducing more hydrophobic function groups to replace the benzamide of DC541. The most cell-potent inhibitor GD562 exhibited over 100-fold selectivity over the other five SAM-dependent protein methyltransferases. Although GD562 showed decreased biochemical inhibition compared to DC541, it demonstrated an over 2-fold increase in cellular inhibition activity on me3-RCC1. In addition, it also inhibited the me3-SET with a similar IC


Relatively little is known about 1 and 2 in adult patients with liver disease. The chemical synthesis of 1 and 2 is therefore described and their quantitation in plasma by ultrarapid chromatography-triple quadrupole mass spectrometry. Plasma concentrations of 1 and 2 were investigated in 25 adult patients with varying degrees of liver cirrhosis with and without hepatocellular carcinoma (HCC). Highly statistically significant correlations (P < 0.0001) were found between severity of liver cirrhosis, determined by the Child–Pugh and MELD scores, with plasma 1 and 2 concentrations, both alone and combined. The presence of HCC did not influence these correlations. Plasma cholic, chenodeoxycholic, deoxycholic, lithocholic or ursodeoxycholic acids, free and as their glycine or taurine conjugates, did not correlate with Child–Pugh or MELD score when corrected for multiple comparisons. These findings demonstrate that plasma levels of fetal bile acids 3-oxachola-4,6-dien-24-oic acid and 7α-hydroxy-3-oxochol-4-en-24-oic acid and likely deteriorating AKR1D1 activity indicate the severity of liver cirrhosis measured by the Child–Pugh and MELD scores.Two 3-oxo-Δ In 2017, cirrhosis caused 440,000 deaths in females and 883,000 in males2. Despite the use of vaccines and therapeutic agents to reduce hepatitis B and C virus infection that contribute to the burden of liver disease, over-nutrition, obesity, metabolic syndrome and alcohol consumption have become principal causes of liver disease3. Hepatic fat overload (steatosis) occurs commonly and can progress to nonalcoholic steatohepatitis (NASH)4 and alcoholic steatohepatitis (ASH)5, in the case of excessive alcohol consumption. Liver disease advances from stage to stage and therefore procedures have arisen for monitoring this progression with the aim of reducing the incidence of end-stage liver disease and HCC4. One such strategy involves the search for biomarkers indicative of disease stage. Mass spectrometry-based metabolomics has proven to be an excellent tool with which to examine premalignant liver disease and its progression to HCC6. It had been reported that the two fetal bile acids 3-oxachola-4,6-dien-24-oic acid (1) and 7α-hydroxy-3-oxochol-4-en-24-oic acid (2), known collectively as 3-oxo-Δ4 bile acids, were elevated in plasma8 and urine8 of HCC patients relative to various control groups, including patients with liver cirrhosis.Over recent decades, the global prevalence of liver disease has continuously risen until it is now one of the leading causes of morbidity and mortality, with liver fibrosis, liver cirrhosis and progression to hepatocellular carcinoma (HCC) on the rise4 bile acids normally attenuates within the first few days after birth9. Elevated 3-oxo-Δ4 bile acids in neonates and children with severe cholestatic liver disease was at first interpreted as being due to an autosomal recessive inborn error of metabolism that affected Δ4-3-oxo-steroid 5β-reductase11 (now known as AKR1D1). The augmentation of fetal bile acids 1 and 2 in urine of infants with hepatobiliary disease was considered to indicate a poor prognosis12. It was also proposed that 3-oxo-Δ4-steroid-5β-reductase reduced activity could have non-genetic origins, such as altered hepatocyte redox state or unusual sensitivity of the enzyme to liver damage. Therefore, it was anticipated that 3-oxo-Δ4 bile acids could be expected to be found in other severe liver diseases10. This proposition has implications for the observation of 3-oxo-Δ4 bile acids in adult patients with liver disease.The excretion of 3-oxo-Δ13. Figure 2) that is formed in the acidic pathway. It is noteworthy that both these species are metabolized by AKR1D1 (Δ4-3-oxosteroid 5β-reductase)13.The synthesis of bile acids from cholesterol proceeds via two alternative pathways, the first of which is the classical “neutral” pathway involving cholesterol 7α-hydroxylation by CYP7A1 with only the final steps entailing creation of the acidic side-chain and leading to cholic acid (CA) and chenodeoxycholic acid (CDCA). In the alternative “acidic” pathway, formation of the acidic side-chain is the first step involving CYP27A1 that finally leads to the synthesis of CDCA14. Defective AKR1D1 activity results in elevated 3-oxo-Δ4 bile acids in neonates11. Whether or not this occurs in adults with liver disease is not known.It has been reported that AKR1D1 hepatic expression in adults decreased with progression of steatosis, fibrosis and inflammation and in type 2 diabetic patients4 bile acids could inform regarding the progression of liver cirrhosis to HCC. Because authentic standards of 1 and 2 are not readily available, we describe here their chemical synthesis from chenodeoxycholic acid. Moreover, quantitation of 1 and 2 has hitherto be achieved using gas chromatography-mass spectrometry (GC–MS) methodologies with complex sample preparation and derivatization. We therefore describe a simple and rapid ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-TQMS) assay for these two fetal bile acids in order to promote further research in this area. Importantly, we investigated 15 liver cirrhosis patients with severity measured by Child–Pugh scores15 of 5–15 (A-C) together with ten cirrhotic patients with HCC and Child–Pugh scores of 5–11. Using the UPLC-TQMS assay based upon authentic standards for 1 and 2, the concentration of these fetal bile acids together with 15 conjugated and free primary and secondary bile acids was determined in plasma from these 25 patients. The findings indicate that plasma fetal bile acids 1 and 2, but not the common bile acids, increase statistically significantly with mounting severity of cirrhosis.We wished to understand if plasma concentrations of 3-oxo-Δ16 from 5 to 15 in these patients. Clinical details of the ten HCC and 15 LC patients are given in Tables 1 and 2 were detected and quantitated in plasma from all 25 cirrhotic patients. In the current investigation, all 25 patients investigated had varying degrees of LC. Examination of their plasma by UPLC-QTOFMS yielded concentrations of fetal bile acids 1 and 2 and we analyzed the findings in relation to both Child–Pugh classes A, B and C together with the Child–Pugh scores from 5 to 15, which represent the severity of the cirrhosis16. Figure 1 and 2. Comparison between Child–Pugh A and Child–Pugh B/C for all cirrhotic patients with and without HCC revealed statistically significant differences for the plasma concentrations of both fetal bile acid 1  and fetal bile acid 2 .Twenty-five patients with LC, comprising 10 with HCC and 15 without, were investigated. The severity of cirrhosis was represented across the full range of Child–Pugh scores1 and 2 were statistically significantly higher in patients with MELD scores ≥ 15 than those with values < 1517  late in the acidic pathway13. Statistical analysis of product/substrate ratios in patient plasma, specifically, CDCA/FBA1, CDCA/FBA2 and CDCA/(FBA1 + FBA2) revealed no statistically significant correlations with Child–Pugh scores (data not shown). Importantly, the presence of HCC did not affect the plasma concentrations of the fetal bile acids, which appeared to be related to severity of cirrhosis as determined by the Child–Pugh criteria. For FBA1, median plasma concentrations with and without HCC were 53.8 and 83.1 ng/mL, respectively (P = 0.50). For FBA2, these values were 72.1 and 85.6 ng/mL, respectively (P = 0.64). Moreover, plasma concentrations of alpha-fetoprotein . AFP values, as expected were statistically significantly higher for patients with HCC (P = 0.007).In addition, the 3-oxo-ΔIn addition to the determination of plasma fetal bile acids, 15 primary and secondary bile acids (free and glycine and taurine conjugated) were determined in patient plasma by UPLC-QTOFMS. Figure Moreover, correlations for the 15 free and conjugated primary and secondary bile acids were also sought using the Spearman rank correlation due to the non-Gaussian distribution of the bile acid and MELD score data. After Bonferroni correction for multiple comparisons, only the taurine conjugated primary bile acids TCA  and TCDCA  correlated statistically significantly. These were stronger correlations than those with the Child–Pugh score). The other 13 bile acids did not correlate with the MELD score with Spearman r values from 0.029 to 0.469 . In addition, an analysis of the 15 bile acids was undertaken in relation to the presence or absence of each of ascites, encephalopathy and esophageal varices. None of these 45 analyses conducted using the nonparametric Mann–Whitney U test produced statistically significantly differences after Bonferroni correction for multiple comparisons.As for the fetal bile acids in Figs. In summary, plasma concentrations of fetal bile acids 1 and 2 alone and combined were highly correlated with cirrhosis severity Figs. . This wa9. However, elevated concentrations of these 3-oxo-Δ4 bile acids have been detected in neonates and children with congenital Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency20. Tentative identification of fetal bile acids 1 and 2 in adults was made in a plasma metabolomics and lipid profiling investigation of HCC patients that used age-matched AML cases as controls. This identification was made on the basis of mass spectroscopic characteristics without authentic chemical standards for 1 and 27. Apart from this report of 20 HCC patients vs. 22 acute myeloid leukemia (AML) controls7, these fetal bile acids had only previously been reported in neonates20.As their name suggests, fetal bile acids occur predominantly in the fetus and are known to decline significantly in the first few days after birth1H NMR and of the two final products 1 and 2 by 1H NMR and high-resolution mass spectrometry. Determination of 1 and 2 in children and neonates with Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency20 has typically been performed using urinary analysis by negative ion fast atom bombardment mass spectrometry (FAB-MS) and gas chromatography-mass spectrometry (GC–MS) in a complex assay that involved hydrolysis of bile acid conjugates in urine after overnight incubation with bile salt hydrolase and double derivatization by methylation and trimethylsilylation11. We describe here for the first time a rapid and simple quantitative UPLC-TQMS assay for the fetal bile acids 1 and 2, which has been utilized to investigate plasma from patients with various degrees of liver cirrhosis, both with and without HCC. This investigation established several unique conclusions. First, plasma concentration of the two fetal bile acids was not related to the presence of hepatic tumors. This could be due to small patient numbers, requiring a larger study to solve this issue. In a previous metabolomic study7, plasma levels of 1 and 2 were upregulated relative to AML but it was unclear if this was due to HCC or LC. Second, plasma concentrations of the fetal bile acids correlated strongly with increasing both Child–Pugh and MELD scores, indicating a relationship between these bile acids and severity of cirrhosis. Third, a further 15 both primary and secondary bile acids and their glycine and taurine conjugates were neither correlated with Child–Pugh classes A, B and C nor with the MELD score, once allowance for multiple comparisons was made. These findings were also unrelated to the presence of HCC. These results point to plasma fetal bile acids as the only indication of severity of cirrhosis known so far among the bile acid family.Because these authentic standards are not commercially available, a detailed account of their chemical synthesis is provided here, together with characterization of the intermediates by AKR1D1 expression by qPCR or AKR1D1 protein by western blotting. This would have permitted us to confirm the role of AKR1D1 in fetal bile acid disposition, which we posited is compromised with increasing severity of cirrhosis.The highlights of this study were that we describe a step-by-step chemical synthesis of these two fetal bile acids, which are not commercially available. In addition, we have described a simple, rapid and quantitative liquid chromatography-tandem mass spectrometry assay for their quantitation in human plasma. We have applied these novel methodologies to the investigation of hepatic cirrhosis. The limitations of our study concern this last component. First, we recruited relatively few patients (n = 25) with HCC (n = 10) and without HCC (n = 15). This group of patients was highly heterogeneous in relation to their complications arising from decompensation, with encephalopathy (n = 7), ascites (n = 8) and esophageal varices (n = 9). Notwithstanding this heterogeneity, clear correlations between each fetal bile acid and both the Child–Pugh score and the MELD score were obtained. A further limitation of this investigation is that liver tissue was not available from the patients and therefore it was not possible to determine 4-3-oxosteroid 5β-reductase (AKR1D1) deficiency, it is tempting to propose that deteriorating liver function may also affect the activity of this hepatic enzyme leading to elevated fetal bile acids as a phenocopy of the inborn error of metabolism. It has been postulated10 that excessive amounts of Δ4-3-oxo bile acids might also be expected to occur in children with severe liver diseases such as hepatitis B and autoimmune chronic active hepatitis but not in more benign chronic cholestatic syndromes such as biliary atresia. In this report, a single patient aged 15 months with a deficiency in fumarylacetoacetase (FAH), known as hereditary tyrosinemia type I that is associated with liver cirrhosis and fatal HCC at a young age21, had the highest ratio of 7α,12α-dihydroxy-3-oxo-4-cholenoic acid  to cholic acid in a series of 25 children and neonates with various liver diseases10. These authors suggested that an altered redox state in hepatocytes or an unusual sensitivity of Δ4-3-oxosteroid 5β-reductase to liver damage might explain their findings. Elevated plasma bile acids, in particular, both free and conjugated CDCA, have been reported to be elevated in cirrhotic patients relative to healthy volunteers22. Total bile acids and total bile acids/cholesterol ratio were found to be elevated in 451 cirrhotic patients compared with 216 non-cirrhotic patients with non-cholestatic chronic HBV infection in China23. An investigation of 32 cirrhotic patients and 27 healthy volunteers examined 12 serum bile acids using UPLC-TQMS and reported that serum concentrations of the conjugated primary bile acids, taurocholic acid (TCA), glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) were positively associated with Child–Pugh classification24. Our own data would appear to corroborate these observations, with uncorrected P-values for nonparametric analyses of variance  of the relationships between plasma TCA, GCA and TCDCA with Child–Pugh class. However, when corrected for false discovery, these relationships ceased to remain statistically significant, as did any relationship between non-fetal bile acids and the MELD score.Because of the large volume of research on fetal bile acids and inborn Δ25. These dramatic findings were of a much higher order than those in our study. It would be of great interest to know how fetal bile acids concentrations were modified in such patients.In a study of alcoholic cirrhosis in heavy alcohol drinkers, striking increases in taurine conjugated bile acids in serum were observed (50- to 130-fold) with more moderate increases in glycine conjugated bile acids (10- to 20-fold). Increased levels of glycochenodeoxycholic acid (GCDCA), TCDCA, GCA, and TCA were positively correlated with disease progression from Child–Pugh A to C26. Perusal of the raw data establishes that the AFP values for our patients are not diagnostic for the presence of HCC. AFP was detected in 9/15 cirrhotic patients without HCC with a median value of 2.6 ng/mL compared with 9.0 ng/mL for those with HCC. AFP is well known to vary in HCC from normal values (< 10 ng/mL) to extremely high values . When used diagnostically, an AFP of 20 ng/mL shows good sensitivity but low specificity and in contrast a value of 200 ng/mL has a high specificity but a sensitivity of only 22%27. It is interesting to propose that deteriorating liver function in the direction of HCC represents an evolving fetal phenotype, particularly when considering the fetal bile acid changes reported here. However, there was a lack of correlation between AFP and fetal bile acid concentrations. In addition, the inborn error of metabolism involving Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency that leads to the accumulation and urinary excretion of fetal bile acids 1 and 219, has not been investigated in relation to increased postnatal serum AFP concentrations. The role of an evolving fetal phenotype in LC that may lead to HCC is an open question.Plasma or serum AFP concentrations are generally regarded as a useful addition to surveillance by ultrasound, CT or MRI imaging for the detection of early HCC and this has been established by a meta-analysis of 32 studies involving 13,367 patients1 and 2? A generalized increased bile acid synthesis, as eluded to above, may result in elevated plasma concentrations of the intermediates 1 and 2. Alternatively, as is the case with the inborn error of metabolism with genetically impaired Δ4-3-oxosteroid 5β-reductase deficiency, AKR1D1 activity may be impaired by an as yet undefined mechanism, leading to worsening cirrhosis. In human liver biopsies taken from 34 obese patients, AKR1D1 mRNA expression was reported to decrease with advancing steatosis, fibrosis and inflammation14. Glucocorticoids, including endogenous cortisol, both downregulate AKR1D1 and are metabolized by it28. A reduced plasma clearance of cortisol in cirrhosis has long been observed29, congruent with a slower metabolic turnover of cortisol in cirrhotic patients30. Taken together, these and other reports may provide an biochemical basis for the association of increased fetal bile acids in patients with advancing cirrhosis.The issue remains how does severity of liver cirrhosis influence the plasma concentration of fetal bile acids All reagents for the chemical syntheses that were conducted in South Korea were purchased from Sigma-Aldrich Korea Ltd. and Tokyo Chemical Industry Co., Ltd.3). Multiplicities in the 1H NMR spectra as described as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br. s = broad singlet. Liquid chromatography-mass spectra (LCMS) were measured in positive electrospray ionization (ESI) mode on a Waters Xevo G2-XS instrument.Product purification was conducted by preparative flash column chromatography using ZEOprep 60 unbonded silica (230–400 mesh). All NMR experiments were carried out using a Bruker Avance III 400 MHz NMR spectrometer equipped with a 5-mm broadband-observed probe head. Chemical shifts were measured relative to residual solvent peaks as an internal standard set to δ 7.26  were conducted as follows: a. Compound 3 , formic acid (11 mL) and 70% perchloric acid  were mixed and the reaction stirred at 55 °C for 1.5 h. To the reaction mixture was slowly added acetic anhydride (9 mL) at 40 °C and then stirred at 55 °C until bubbling ceased. The mixture was then cooled to RT and slowly added to water and extracted with ethyl acetate. The separated organic phase was washed with brine, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified using flash column chromatography  to afford the 3,7-diformyl intermediate . b. To the 3,7-diformyl intermediate  in acetone (4 mL) was slowly added 0.2 M sodium hydroxide  and the reaction mixture stirred at RT for I h, neutralized to pH 7 with acetic acid, diluted with water and extracted with dichloromethane. The separated organic phase was washed with brine, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified using flash column chromatography  to afford compound 4 .Procedures a and b for the synthesis of 5 was conducted as follows: To compound 4  in acetone (11 mL) was slowly added Jones’ reagent (0.3 mL) at 4 °C and the reaction stirred at this temperature for 2 h, then at RT for 1 h, whereupon methanol (0.6 mL) was slowly added to the reaction mixture to form a suspension, which was filtered and washed with acetone. The combined filtrate was concentrated under reduced pressure and the resulting residue purified using flash column chromatography  to afford compound 5 .Procedure c for compound 6 was conducted as follows: To compound 5  and p-toluenesulfonic acid  in DMF (5 mL) was slowly added bromine  in DMF (1 mL) at RT and stirred for 20 h. The mixture was then slowly added to water and extracted with ethyl acetate. The separated organic phase was washed with brine, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified using flash column chromatography  to afford compound 6 .Procedure d for compound 1 and 7 was conducted as follows: A solution of semicarbazide.HCl  and sodium acetate  in water (1 mL) was added dropwise to compound 6  in acetic acid (8 mL) and the mixture stirred at 60 °C for 0.5 h, then stirred at RT for 1 h to form a precipitate. This solid was filtered, washed with pure water and used without further purification. This solid, acetic acid (9 mL), pyruvic acid (0.9 mL) and water (2.1 mL) were stirred at RT for 20 h. Precipitated solid was filtered off and the filtrate extracted with ethyl acetate, which was washed with brine and dried over anhydrous MgSO4, filtered and evaporated under reduced pressure. The resulting residue was purified using flash column chromatography  to afford compound 1  and compound 7 .Procedure e for compounds 2 was conducted as follows: Compound 7  was dissolved in 0.5 M sodium hydroxide (10 mL) and stirred at RT for 1.5 h. The reaction mixture was acidified to pH 1–2 with 1 M HCl and extracted with ethyl acetate. The combined organic phase was washed with brine, dried over anhydrous MgSO4 and evaporated under reduced pressure. The resulting residue was lyophilized without further purification to afford compound 2 .Procedure f for compound 1 and 2 were as follows:(R)-4--10,13-dimethyl-3-oxo-2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (1). m.p. 198–200 °C. 1H NMR  δ 6.15 , 6.07 , 5.55 , 2.60–2.47 , 2.32–2.08 , 2.03–1.92 , 1.90–1.82 , 1.80–1.68 , 1.66–1.57 , 1.54–1.44 , 1.43–1.28 , 1.26–1.10 , 1.06 , 1.00 , 0.91–0.83 , 0.71 , 0.68–0.61 .; LCMS (ESI): Exact mass calculated for C24H34O3 [M + H]+ 371.2586, found 371.2603 (Δm/z = 4.6 ppm).(4R)-4--7-hydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (2). m.p. 228–230 °C (literature32 227–229 °C). 1H NMR  δ 5.64 , 3.83 , 2.62–2.53 , 2.46–2.05 , 2.02–1.91 , 1.91–1.78 , 1.75–1.61 , 1.59–1.18 , 1.16–1.03 , 0.87 , 0.67 ; LCMS (ESI): Exact mass calculated for C24H36O4 [M + H]+ 389.2692, found 389.2706 (Δm/z = 3.6 ppm).Spectral data for synthetic fetal bile acids Spectral data for the intermediates 4, 5, 6 and 7 are given in the 1 and 2 eluted at 1.08 and 0.82 min, respectively. For fetal bile acid 1, the MRM used was 369.3 > 325.4 with a dwell time of 0.069 s, a cone voltage of 100 V and a collision energy of 23 eV. For fetal bile acid 2, the MRM used was 387.3 > 369.4 with a dwell time of 0.069 s, a cone voltage of 100 V and a collision energy of 19 eV. The internal standard (IS) employed was 3β,7α-dihydroxy-5-cholesten-26-oic acid (DHCA) added at a concentration of 500 ng/mL, which eluted at 0.43 min. The MRM for the IS used was 401 > 331 with a cone voltage of 60 V and a collision energy of 20 eV. Peak area ratios to the internal standard were used for construction of calibration curves. For fetal bile acid 1 (FBA1), the calibration curve was linear . For fetal bile acid 2 (FBA2), the calibration curve was also linear . Precursor and product ions were similar to a published method but with lower collision energies33. The advantage of this assay is the short retention times (< 1.2 min) for 1 and 2, compared with 33.2 and 29.0 min, respectively, as reported earlier33.Human plasma (25 μL) was protein precipitated with methanol (475 μL) and centrifuged for 3 min at 25,000 g. Resultant supernatant (100 μL) was diluted with deionized water (100 μL) and 10μL injected into the UPLC-TQMS system, which comprised an Acquity I-Class UPLC and a Xevo-TQ-S Micro mass spectrometer (Waters). UPLC separation was achieved on a CORTECS T3 2.7 μm (2.1 × 30 mm) analytical column with a flow rate of 1.3 mL/min. Mobile phases A and B were 0.01% aqueous formic acid and isopropanol/acetonitrile (50:50 v/v) containing 0.01% formic acid, respectively. After an initial 0.1 min at 30% B, the bile acids were eluted with a gradient of 30–65% B over 0.7 min, followed by a 0.9 min column wash with 98% B and re-equilibrated to initial conditions. The analytical column was maintained at 60 °C. Multiple Reaction Monitoring (MRM) analyses were performed in negative electrospray ionization mode. Ion source temperature and capillary voltage were held constant at 150 °C and 2.0 kV, respectively. The cone gas flow was 50 L/h and desolvation temperature was 650 °C. Fetal bile acids g for 3 min. Resultant supernatant (100 μL) was diluted with deionized water (100 μL) and 10 μL injected into the UPLC-TQMS system (see above). UPLC separation was achieved on a CORTECS T3 2.7 μm (2.1 × 30 mm) analytical column with a flow rate of 1.3 mL/min. Mobile phases A and B were 0.01% aqueous formic acid containing 0.2 mM ammonium formate and isopropanol/acetonitrile (50:50 v/v) containing 0.01% formic acid and 0.2 mM ammonium formate, respectively. After 0.1 min at 20% B, the bile acids were eluted with a gradient of 20–55% B over 0.7 min, followed by a 0.9 min column wash with 98% B and re-equilibrated to initial conditions. The analytical column was maintained at 60 °C. MRM analyses were performed as above. Retention times together with MRM transitions, cone voltages and collision energies are shown in Supplementary Table 34. However, in the case of CDCA, the primary bile acid most closely related to FBA1 and FBA2, a calibration curve was constructed as for the fetal bile acids above. For CDCA, the calibration curve was linear .Human plasma (100 μL) was protein precipitated with methanol (400 μL) and centrifuged at 25,000×16 for the HCC patients  were A (5/10), B (4/10) and C (1/10) and for the LC patients were A (12/15), B (2/15) and C (1/15), corresponding to Child–Pugh scores16 of 5–11 for HCC and 5–15 for LC. MELD scores for the HCC patients ranged from 6 to 18 and for the LC patients ranged from 6 to 27. Peripheral venous blood was drawn into heparinized tubes for each patient and spun at 1300 g to prepare plasma, which was aliquoted and frozen at − 80 °C prior to shipment to Switzerland by courier on dry-ice. Plasmas were stored at − 80 °C in Bern before forwarding of aliquots by courier on dry-ice to Wilmslow, UK where they were analyzed by UPLC-TQMS for fetal bile acids 1 and 2, together with 15 free and conjugated primary and secondary bile acids as described above.Patients were recruited from the Octavian Fodor Institute for Gastroenterology and Hepatology Hospital, Cluj-Napoca, Romania. All patients gave their written informed consent prior to study inclusion. The study was performed in accordance with the 1975 Declaration of Helsinki and approved by the institutional review board of the Regional Institute of Gastroenterology and Hepatology “Prof. Dr. Octavian Fodor”, Cluj-Napoca, Romania . All patients included in this study were hospitalized, having been admitted for liver cancer screening, because of a first decompensation event , because of newly diagnosed focal liver lesions on a background of liver cirrhosis or because of referral for treatment . HCC patients (n = 10) were consecutively enrolled into the study. During the same period, consecutive patients with liver cirrhosis (LC) but no HCC (n = 15) were included. The diagnosis of HCC was made according to non-invasive criteria or based on histology in patients with atypical lesions on a background of liver cirrhosis. LC patients included those on the first diagnosis of cirrhosis based on ultrasound, laboratory data, Fibroscan, CT/MRI and/or hepatic venous pressure measurement with liver biopsy or patients who were known with a history of liver cirrhosis and presented with cirrhosis decompensation . The exclusion criteria were: (1) previous or concurrent cancer; (2) active infections other than chronic hepatitis B or hepatitis C virus; (3) history of organ allograft; (4) pregnancy or breastfeeding; and (5) refusal to sign for informed consent. Clinical details of HCC patients and LC patients are given in Tables Statistical analysis was performed using GraphPad Prism 9.1.0  by nonparametric statistics because of the general non-Gaussian distribution of the data. Comparisons between two groups of data were analyzed using the Mann–Whitney U test and between three or more groups using the Kruskal–Wallis test. Correlations between variables were made by Spearman’s rank correlation. All values are expressed as medians. P < 0.05 was considered statistically significant. The Bonferroni correction was used when multiple comparisons were made.Supplementary Information.


The semiconductor was found to be metastable, i.e., thermodynamically unstable, and to slowly oxidize at its surfaces upon heating in air, yielding CuO as nano-sized islands. However, the bulk crystalline structure was maintained, with up to 50% Cu(I)-vacancies and a concomitant oxidation of the Cu(I) to Cu(II) cations within the structure. Thermogravimetric and magnetic susceptibility measurements showed the formation of increasing amounts of Cu(II) cations, according to the following reaction: Cu3Ta7O19 + x/2 O2 → Cu(3−x)Ta7O19 + x CuO (surface) (x = 0 to ~0.8). With minor amounts of surface oxidation, the cathodic photocurrents of the polycrystalline films increase significantly, from <0.1 mA cm−2 up to >0.5 mA cm−2, under visible-light irradiation  at an applied bias of −0.6 V vs. SCE. Electronic structure calculations revealed that its defect tolerance arises from the antibonding nature of its valence band edge, with the formation of defect states in resonance with the valence band, rather than as mid-gap states that function as recombination centers. Thus, the metastable Cu(I)-containing semiconductor was demonstrated to possess a high defect tolerance, which facilitates its high cathodic photocurrents.A p-type Cu This results in a shortened energetic distance to the conduction band of an early transition-metal or main group oxide, as found in several previous examples, such as CuRhO2 [2WO4 [3 [3VO4 [p-type semiconducting oxides have previously been reported, e.g., Cu2O [2O4 [p-type semiconductors.Recently, many mixed-metal oxide semiconductors containing a Cu(I) cation have emerged as promising ectrodes ,2. Incors CuRhO2 , Cu2WO4 [2WO4 [3 , Cu3VO4 [3 [3VO4 , and othg., Cu2O ,8 and Cau2O [2O4 ,10, many2O is reacted with M2O5  or M’O2 (M’ = Ti or Hf), nearly all of the ternary Cu(I)-containing oxides are thermodynamically unstable, i.e., metastable, with respect to decomposition to simpler oxides. For example, when Cu2Ta4O11 is heated in a vacuum, the following decomposition occurs: Cu2Ta4O11 → Cu2O + 2 Ta2O5. This reaction, and other alternative decomposition pathways, have been observed in all Cu(I)-containing niobates, tantalates, and vanadates [2Ta4O11 is calculated to be metastable by ~0.04 eV atom−1, and thus its decomposition reaction is thermodynamically favorable. Most known Cu(I)-semiconductors are calculated to be metastable, as tabulated within the Materials Project database [Low-temperature synthetic techniques, such as flux-mediated or hydrothermal ,12,13, hanadates ,16,17,18database . The reldatabase . This leg~1.3 to 2.6 eV) compared to most other metal oxides, with high attainable photocathodic currents (up to ~3–5 mA/cm2) in the form of polycrystalline films. The latter is enhanced by mild oxidation of their surfaces in air. For example, recent studies on several Cu-containing semiconductors, including CuNb3O8 [3VO4 [5Ta11O30 [2O4 [Cu(I)-containing semiconductors possess advantageously smaller band gaps  and partial oxidation of the Cu(I) to Cu(II) cations, and yet with maintenance of its bulk crystalline structure. Its electronic structure was probed by density functional theory calculations, which revealed features that lead to its high defect tolerance. Thus, the metastable nature of this Cu(I)-containing semiconductor is demonstrated to be closely related to its ability to tolerate a high concentration of defect sites, leading to large cathodic photocurrents.Presented herein is the flux synthesis and investigation of the photoelectrochemical properties of polycrystalline films of the metastable, Cu(I)-containing, Cu3Ta7O19 compound was prepared with high purity using CuCl-flux mediated synthesis. The use of a CuCl flux is key to preparing this compound in high purity, as it is calculated to be metastable and to decompose to a mixture of Cu2O and Ta2O5 by 0.032 eV atom−1 [3Ta7O19 crystallizes in the hexagonal P63/m space group, with a structure type that has been described previously [7 pentagonal bipyramids. The layers of pentagonal bipyramids, i.e., the TaO7 in 3O8 [The powder XRD data, eviously . The str7 in 3O8 .3Ta7O19, the crystallites were heated in air at temperatures spanning the range of 350 °C to >550 °C. Shown in 3Ta7O19 crystallites with highly faceted and smooth surfaces, with a size distribution between ∼5 to 20 μm. After heating these in air at 350 °C for 60 min, 450 °C for 60 min, or at 550 °C for 60 min, increasing amounts of surface nano-islands were found, as shown in 3O8, Cu3VO4, Cu5Ta11O30, and Cu2WO4 [ab-plane of the structure, as investigated by electron microscopy and described previously for the related Cu5Ta11O30, which contains structurally similar layers of pentagonal bipyramids [To probe the bulk and surface-level thermal stability of Cud Cu2WO4 ,17; i.e.3Ta7O19 were performed after heating in air, in order to understand the bulk changes in the crystalline structure that accompany the surface oxidation. Shown in x)(3−Ta7O19 (x = 0.34(1)), or with a copper-deficiency of ~11.3%, as listed in 2Ta4O11 (33% vacancies) and Cu5Ta11O30 (16.6% vacancies) [Refinements of the unit cell or full crystalline structure of Cucancies) ,25. Thus3Ta7O19 + x/2 O2 → Cu(3−x)Ta7O19 + x CuO(surface). With the extrusion of Cu(I) cations from the structure and oxidation at the surfaces, an equal number of Cu sites within the bulk are also oxidized, as given by simple charge balancing: CuI(3–2x)CuIIxTa7O19. Temperature-dependent magnetic susceptibility measurements were taken on Cu3Ta7O19 before and after heating in air from 250 °C to 450 °C. Listed in B with no heating, to 0.216μB, 0.452μB, and 0.917μB after heating in air to 250 °C, 350 °C, and 450 °C, respectively. Accounting for both the surface and bulk Cu(II) cations, these values correspond to %Cu(II) cations  in the bulk Cu(3−x)Ta7O19, ranging from ~10.4% (x = 0.015) to 26.1% (x = 0.39) at 50 °C, and to 53.0% (x = 0.8), respectively. This analysis is consistent with the refined Cu-site vacancy from the Rietveld analysis, with x = 0.34(1) after heating to 350 °C in air, and supports the oxidation of Cu(I) to Cu(II) cations within the structure and at its surfaces according to the above reaction. Full oxidation eventually leads to CuTa2O6, as shown in the powder XRD data in The ion diffusion and phase segregation of Cu(I) cations out of the structure, and their oxidation to CuO at the surfaces, can be represented by the following reaction: Cu3Ta7O19 had a band gap of ~2.5 eV, 3Ta7O19 semiconductor were deposited onto FTO slides using a previously reported drop-cast method and annealed in vacuum at 500 °C. All films showed a p-type semiconducting behavior, with an increasing photocathodic current with negative applied biases. Higher photocurrents were found for films that had been heated in air at increasing temperatures to slightly oxidize their surface. Shown in −2), and this increased after heating at 350–550 °C in air for 20 min  under chopped visible light irradiation at 500 mW cm−2 and pH = 6.3. Chronoamperometry data taken at −0.25 applied bias, (3−x)Ta7O19, corresponding to the composition with the highest number of Cu-site vacancies. The polycrystalline film of Cu3Ta7O19 was also heated to 450 °C at increasing time intervals of 20, 40, and 60 min, to investigate the impact of increasing thermal oxidation. The photocathodic currents ranged from <0.1 mA cm−2 at 0 V applied bias to ~1.0 mA cm−2 at −0.6 applied bias, and generally increased by ~50% after heating from 20 min to 60 min, as shown in Tauc plots of the UV-Vis diffuse reflectance data show that Cu(3−x)Ta7O19 films. This result is surprising given the high concentration of Cu-site vacancies, of up to 50%, that formed when heating in air at 450 to 550 °C. Prior work on Cu(I)-containing semiconductors has shown that the phase segregation of CuO at the surface can result in a favorable band-energy offset to drive more efficient charge separation, such as in Cu(3−x)VO4 and CuBi2O4 [3Ta7O19, its band edge energies were determined from Mott–Schottky measurements that yielded a flat band potential of +0.36 V (versus SCE at pH = 6.3). This gave band edge energies of −1.57 V and +0.73 V for the conduction and valence band edges, respectively. The conduction band edge is therefore located at a significantly more negative potential than for CuO, which occurs at about −0.8 V (+/− 0.1 V) at pH ~ 6.0 versus SCE [3Ta7O19 to CuO, followed by the reduction of protons to dihydrogen at the surfaces of the latter, as illustrated in 3Ta7O19 is located at a more negative potential than in CuO, inhibiting the concomitant hole migration to the surfaces of CuO. Thus, a favorable type-II band offset is formed between p-type CuO and Cu3Ta7O19, as shown in The oxidation of Cu(I) to Cu(II), occurring both at the surface and in the bulk, yielded much higher photocurrents for the Cu CuBi2O4 ,21. For rsus SCE ,26. The 3Ta7O19 is perhaps surprising, given the potential impact of defect sites functioning as charge recombination centers. In semiconductors, defects can function as deleteroius recombination centers if they introduce mid-gap states within the electronic structure. However, if the formation of mid-gap states can be avoided or suppressed, then the semiconductor is described as being ‘tolerant’ to the formation of defects and can still function as an efficient photoelectrode [The increased photocurrent with higher concentrations of Cu(I)-vacancies in Culectrode . It is u(3−x)Ta7O19, Cu-site vacancy defects are shown to occur in the bulk (up to x = 0.8) as a result of their extrusion from the structure and oxidation at the surfaces to CuO. Electronic structure calculations were performed using density-functional theory methods, to investigate the energies and impact of the Cu-site vacancies, as well as the atomic contributions to the conduction and valence band edges, as illustrated in (3−x)Ta7O19 with 8.3% Cu-site vacancies, i.e., x = 0.33. The electron densities at the conduction and valence band edges are shown to stem from the filled Cu 3d10 and empty Ta 5d0 orbitals, respectively, as found previously for the idealized structure without Cu-site vacancies [10 orbitals. This is consistent with the partial oxidation of Cu(I) to Cu(II) cations, as was found experimentally. The electron densities at the conduction and valence band edges remain unchanged, as a result of the introduction of Cu-site vacancies shown in (3−x)Ta7O19 structure shows a high defect tolerance, with its high concentrations of Cu-site vacancies yielding increasing cathodic photocurrents, due to the formation of CuO at the surfaces.In Cuacancies . The int3Ta7O19 semiconductor was prepared by a flux-mediated synthetic route and investigated for its photoelectrochemical properties. Upon heating in air, the Cu(I) cations slowly oxidized to Cu(II) at the surfaces and in the bulk, as probed by electron microscopy and magnetic susceptibility measurements. In the form of polycrystalline films, increasing cathodic photocurrents of up to >0.5 mA cm−2 (under visible-light irradiation) are observed after heating in air from 350 °C to 550 °C, from 20 to 60 min. These increased photocurrents originate from the formation of a type-II band offset between Cu(3−x)Ta7O19 and CuO at the surfaces, which can facilitate an enhanced charge separation. Furthermore, electronic structure calculations show that these high photocurrents were facilitated by the defect tolerance of the structure for Cu-site vacancies and the absence of mid-gap energetic states, which could potentially function as recombination centers.A metastable p-type Cu2O5 , Cu2O , and CuCl . For the photoelectrochemical measurements, the reagents Na2SO4 , NaOH , and deionized water were also used.The reactants were reagent grade and used as received for Ta3Ta7O19, with the low melting CuCl (m.p. = 426 °C) salt as the flux. Stoichiometric amounts of Cu2O and Ta2O5 were finely ground together within a mortar and pestle for 30 min inside an Ar-filled glovebox. To this, the CuCl salt flux was added in a 10:1 molar ratio of salt:product and the powder was loaded into a previously cleaned quartz tube. After sealing under vacuum, the reaction vessel was heated inside a muffle furnace to 800 °C for 26 h and allowed to radiatively cool to room temperature. The products were washed with 3M NH4OH in order to remove the CuCl flux, and yielding a bright, yellow-colored powder that was determined by powder X-ray diffraction to be Cu3Ta7O19 in high purity. To investigate the decomposition of Cu3Ta7O19 upon heating, samples of the high-purity powder were placed into an alumina crucible and heated in air in a muffle furnace in intervals from 350 °C up to 750 °C for heating times from 20 min to 3 h.A flux-mediated synthetic approach was used for the preparation of Cu1 radiation (λ = 1.54056 Å) generated from a sealed tube X-ray generator . A sample mass of ~50 mg was dispersed between two pieces of Scotch tape and fixed into a rotating sample holder. A curved position sensitive detector was used in transmission mode to measure the XRD diffractograms, with exposure times from 30 to 90 min. The lattice constants of the bulk powders were refined using the Rietveld method, using whole pattern fitting within Jade 9 software [3Ta7O19 and for the product after it was heated in air, in intervals from 350 °C to 750 °C.An Inel X-ray diffractometer was used to take powder X-ray diffraction (XRD) data, with Cu Kαsoftware . The uni3Ta7O19 compound was performed after oxidation in air at 350 °C for 60 min. Powder XRD data were collected on a Philips X-pert diffractometer using Cu Kα radiation at room temperature. An angular range of 10° ≤ 2θ ≤ 110° was collected with a step width of 0.017°. The structural refinement proceeded by the Rietveld method within the JANA2000 software package [3Ta7O19 [3/m (no. 176) with lattice constants a = 6.2278(1) Å and c = 20.1467(3) Å and V = 672.7(1) Å3. Its crystalline structure consists of two symmetry-unique Ta sites, one Cu site, and five oxygen sites. Refinement of the background and the peak shape parameters proceeded first, followed by zero shift parameter and the dimensions of the unit cell, refining to a = 6.2403(1) Å and c = 20.0835(3) Å and V = 677.30(2) Å3. The atomic coordinates were refined next, starting with the heavier Ta and Cu atoms, and then the O atoms. Lastly, the Cu-site occupation was allowed to refine and it converged to an occupancy of 0.887, giving a refined composition of Cu2.67Ta7O19 that is consistent with the contracted unit cell volume (by −0.06%) and ~11% Cu-site vacancies. The final refined R and wpR factors converged to 5.61% and 9.45%, respectively. Selected refinement parameters and interatomic distances are given in the A full structural refinement of the Cu package . A Legen[3Ta7O19 , crystal4 powder was used as the reference background. In a typical measurement, ~30 mg of powder was mixed with the BaSO4 reference powder and mounted onto a flat sample holder. The holder was then placed into the external window of the integrating sphere. As described previously [F(R)hν)n vs. (hν) to estimate the direct (n = ½) and indirect (n = 2) bandgap energies. Scanning electron microscopy (SEM) images were taken on a field-emission scanning electron microscope to characterize the particle morphologies, surfaces features, and qualitative chemical compositions. The thermal stability and reactivity of the samples were measured as a function of temperature and the data were analyzed as the % weight change of the sample versus temperature.UV-Vis diffuse reflectance spectra were collected using an integrating sphere within the wavelength range of 200 nm to 1300 nm. A pressed BaSOeviously , the ref3Ta7O19 were prepared on TEK-15 fluorine-doped tin oxide (FTO) slides. The slides were first sonicated in deionized water, followed by ethanol and acetone for 30 min each. On the conducting side, an ~1 cm2 area was taped off using Scotch 3M tape and powdered samples were deposited using drop casting and doctor blade techniques, according to previous reports [2SO4 solution as electrolyte at a pH of ~6.3. Linear-sweep cyclic voltammograms were taken at an applied bias range of 0.2V to −0.6V under chopped irradiation from a high pressure Xe lamp for each film. The arc lamp was equipped with infrared and ultraviolet photon cutoff filters, to give an output wavelength range of 1000 nm to 420 nm at an irradiant power density of ∼500 mW/cm2. Chronoamperometric measurements were also taken at a constant applied bias of −0.25 V, as a gauge of the photocurrent decay versus time.Polycrystalline films of Cu reports ,17. A wa(3−x)Ta7O19 structure, as implemented in the Vienna Ab-Initio Simulation Package  [2.67Ta7O19 were utilized in the calculations. A Cu-site vacancy concentration of ~8.3% was modeled using a 2 × 2 × 1 superstructure, which enabled a random distribution of 1 Cu-site vacancy per four unit cells of Cu3Ta7O19. Perdew–Burke–Ernzerhof functionals were employed in the calculations within the generalized gradient approximation [k-points within the Brillouin zone [Plane-wave density functional theory was utilized to perform electronic band-structure calculations on the geometry-optimized and Cu-deficient Cuer. 4.6) . The refximation . The Monuin zone .