HD2A and HD2B caused hyperacetylation of the DOG1 locus and promoted the expression of DOG1 during seed maturation and imbibition. Knockout of DOG1 could rescue the seed dormancy and partly rescue the disturbed development phenotype of hd2ahd2b. Transcriptomic analysis of the hd2ahd2b line shows that many genes involved in seed development were impaired. Moreover, we demonstrated that HSI2 and HSL1 interact with HD2A and HD2B. In sum, these results suggest that HSI2 and HSL1 might recruit HD2A and HD2B to DOG1 to negatively regulate DOG1 expression and to reduce seed dormancy, consequently, affecting seed development during seed maturation and promoting seed germination during imbibition.Seed dormancy is a crucial developmental transition that affects the adaption and survival of plants. Arabidopsis DELAY OF GERMINATION 1 (DOG1) is known as a master regulator of seed dormancy. However, although several upstream factors of DOG1 have been reported, the exact regulation of DOG1 is not fully understood. Histone acetylation is an important regulatory layer, controlled by histone acetyltransferases and histone deacetylases. Histone acetylation strongly correlates with transcriptionally active chromatin, whereas heterochromatin is generally characterized by hypoacetylated histones. Here we describe that loss of function of two plant-specific histone deacetylases, HD2A and HD2B, resulted in enhanced seed dormancy in Arabidopsis. Interestingly, the silencing of  DOG1 encodes a nuclear protein with unknown biochemical function and is mainly expressed in seed (DOG1 expression. DOG1 expression and increased seed dormancy. This might be triggered by increased expression of transcription factor (TF) bZIP67 which can bind to the DOG1 promoter (DOG1 expression is regulated by epigenetic regulators. Histone demethylases LDL1/LDL2 and histone methyltransferases KRYPTONITE (KYP)/SUVH4/SUVH5 repress DOG1 during seed maturation , and histone acetylation  and HSI2-LIKE1 (HSL1) play also critical roles during plant reproduction and seed germination . HSI2 an of DOG1 . Additio of DOG1 .cis-trans peptidyl-propyl isomerases  or mutants in the Col-0 background were used in all experiments. The T-DNA insertion lines GABI_355H03 (hd2a), Sail_1247_A02 (hd2b), Salk_039784 (hd2c), GK_379G06 (hd2d), and SM_3_20886 (dog1-4) were described previously . After placing the tubes on ice, 300 µl homogenization buffer  was added and the samples were again homogenized for 30 sec. The supernatant was transferred to 1.5 ml microcentrifuge tubes and centrifuged for 10 min at 25,000g and 4°C to remove cell debris. The protein concentration of the supernatant was determined according to Bradford (2) was added. After incubation for 2 h at 37°C, 10 mg/ml trypsin and 1 µ M TSA in 60 µl, HDA stopping buffer  was added per well and fluorescence output, representing HDA activity, was measured after 20 min incubation at 30°C at λ 380nmExcitation and 440nmEmission.Measurements of HDA activity of r et al.  and , the supernatant was removed and total protein was extracted by re-suspending and disrupting the protoplast with 1 ml of extraction buffer . After gentle shaking for 1 h at 4°C, the sample was centrifugated at 12,000g for 10 min. To purify GFP-tagged proteins, the supernatant was incubated with 25 μl GFP-Trap agarose beads  at 4°C overnight by gentle rotation. After washing with extraction buffer four times, proteins were eluted with 50 μl 2x SDS sample buffer and analyzed by immunoblotting using an anti-Myc antibody.For Co-IP analysis, the hd2ahd2b seeds and from 10 d old 35Spro: HD2B-GFP seedlings. The seeds were imbibed at room temperature under the dark, and at that time point, no germination was visible. About 1 g of imbibed seeds and 2 g of seedlings were cross-linked in cross-linking buffer  by vacuum infiltration for 1 h and 10 min, respectively. Cross-linking was stopped by adding glycine to an end concentration of 0.125 M and additional vacuum infiltration for 5 min. The cross-linked plant materials were washed two times with ice water, dried with paper towels, and ground in liquid nitrogen to a fine powder. The chromatin was extracted with 20 ml extraction buffer  by gentle shaking for 20 min and pelleted by centrifugation at 4000g for 25 min at 4°C. The pellet was washed with nuclei washing buffer , re-suspended in 600 μl nuclei sonication buffer  and the chromatin was sheared to 200–1,000 bp by sonication. the nuclei were transferred into 1.5 ml Bioruptor Microtubes (Cat No. C30010016), and 15 cycles with 30 sec ON/OFF was used with Bioruptor® Pico ultrasonic bath and Covaris E220 Evolution. After centrifugation for 10 min at 12,000g and 4°C the supernatant was directly used for immunoprecipitation with specific antibodies. For H3K9ac, H4K5ac, and H4ac analysis, the antibodies  were coupled to the magnetic protein G beads by incubating at 4°C on a rotation platform overnight. Afterward, 100 μl sonicated chromatin was mixed with antibody-magnetic protein G beads and incubated overnight at 4°C on a rotating platform. The beads were sequential washed with low salt buffer , high salt buffer , LiCl buffer , and TE buffer . And the beads were washed twice with each buffer. Finally, the chromatin was eluted with 400 μl of elution buffer  by incubation for 20 min at 65°C, and the chromatin de-crosslinking was performed at 65°C for over 6 h after adding 16 μl of 5 M NaCl. After being treated with proteinase K and RNase, DNA was purified by phenol-chloroform method, eluted with dH2O, and quantified for qPCR. For anti-GFP analysis, the GFP-Trap agarose beads were used instead of antibody-coupled magnetic protein G beads, and analysis was performed with the same procedure as above.The ChIP-qPCR assay was performed as previously described . The chrHD2A, HD2B, HSI2, and HSL1 (without stop codon) were transferred into the pDONOR221 vector by Gateway Cloning and subsequently shifted into the pBiFCt-2in1-NN vector (LR reactions) according to the described . The residue was re-dissolved in 200 μl of 50% methanol and filtered through a 0.22 μm filter for sample loading. For each sample, 100 μl methanol solution was subjected to LC-MS/MS analysis. ABA  and GA3  were used as authentic reference standards. All determinations were performed in triplicate.The endogenous ABA and GA3 contents were measured with Agilent 1290 Infinity II-6470 triple quadrupole LC/MS/MS System according to the previously reported method  with minhd2a, hd2b, hd2c, and hd2d  and reaching the highest expression level in 12 h imbibed seeds  or HD2B-GFP (pHD2B: HD2B-GFP) and the two HD2B overexpression lines HD2B-OE9, HD2B-OE14. DOG1 transcription level in the pHD2B: HD2B-GFP line was comparable to that of WT seeds  is an essential regulator of seed dormancy . The graDOG1 expression in hd2ahd2b during seed maturation and imbibition. We observed that the DOG1 mRNA accumulation decreased from 12 DAP until seed maturation, which is consistent with the reported data in the Col-0 background  germination. Interestingly, this reduced germination phenotype is restored in the complementation line pHD2B: HD2B-GFP and partially in pHD2A: HD2A-GFP. Although the dog1 mutant and the HD2B overexpression lines HD2B-OX9 and HD2B-OX14 showed reduced expression of DOG1 and a similar percentage of germination as WT seeds after 2 days of incubation, the percentage of germination of WT is slightly delayed in comparison to that of dog1 and both HD2B overexpression lines  on 24 h imbibed seeds of WT and hd2ahd2b using specific anti-H3K9ac, anti-H4ac, and anti-H4K5ac antibodies. A 103 bp promoter region P1, 1064 bp upstream of the transcription start site (TSS), and a 189 bp coding region P2, 157 bp downstream of TSS, were amplified with specific primers  and CURLY LEAF (CLF) for consequent deposition of H3K27me3 marks at DOG1 locus   . In seedlings, we found, that 1720 and 772 genes were up- and down-regulated in hd2ahd2b, respectively  analysis of ten days old WT and ectively . Intereshd2ahd2b than up-regulated (~520)  in DOG1 locus to regulate seed development and germination by repressing DOG1.The plant-specific histone deacetylase subfamily HD2s plays multiple functions during plant development by acting as a transcription repressor . In thisHD2A single ko-mutant has an elevated DOG1 expression level  and stronger dormancy phenotype in comparison to the corresponding single mutants  are essential for seed dormancy and germination . DOG1 plDOG1 is essential during seed development, maturation, and storage , accession number PRJEB62044.The RNA sequencing data presented in the study are deposited in the ENA repository (YH and CL conceived research plans; CL, CB, GH, and RK supervised the experiments; YH, SPC, CW, and PH performed the experiments; YH, CL, EG, SPC, GH, and RK designed the experiments and analyzed the data; YH and CL wrote the article with contributions of all the authors; EG, CB, and JD reviewed and edited the text. CL agrees to serve as the author responsible for contact and ensures communication. All authors contributed to the article and approved the submitted version.


Pulmonary fibrosis is a progressive lung disease characterized by macrophage activation. Asbestos-induced expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (NOX4) in lung macrophages mediates fibrotic progression by the generation of mitochondrial reactive oxygen species (ROS), modulating mitochondrial biogenesis, and promoting apoptosis resistance; however, the mechanism(s) by which NOX4 localizes to mitochondria during fibrosis is not known. Here, we show that NOX4 localized to the mitochondrial matrix following asbestos exposure in lung macrophages via direct interaction with TIM23. TIM23 and NOX4 interaction was found in lung macrophages from human subjects with asbestosis, while it was absent in mice harboring a conditional deletion of NOX4 in lung macrophages. This interaction was localized to the proximal transmembrane region of NOX4. Mechanistically, TIM23 augmented NOX4-induced mitochondrial ROS and metabolic reprogramming to oxidative phosphorylation. Silencing TIM23 decreased mitochondrial ROS and oxidative phosphorylation. These observations highlight the important role of the mitochondrial translocase TIM23 interaction with NOX4. Moreover, this interaction is required for mitochondrial redox signaling and metabolic reprogramming in lung macrophages. Pulmonary fibrosis is a chronic fibrotic lung disease with high morbidity and mortality . EnvironMacrophages contribute to asbestosis by producing profibrotic factors, collagen synthesis substrates, and generate high levels of mitochondrial reactive oxygen species (ROS) . Mitocho2O2 (Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 4 (NOX4) is an enzyme of the family NADPH oxidases. NOX4 is constitutively active having the primary function of ROS generation in the mitochondria. In the basal state, NOX4 resides in mitochondria in macrophages. NOX4 catalyzes the reduction of oxygen to superoxide anion-free radicals as well as H2O2 , 9. In a2O2 , 11. NOX2O2 . AlthougHere, we found that NOX4 is present in the mitochondrial matrix and that the import requires direct interaction with TIM23. Because one major role of NOX4 is the generation of mitochondrial ROS, silencing TIM23 abrogated NOX4-mediated ROS production in macrophages. We and others have shown that mitochondrial ROS induced metabolic reprogramming to oxidative phosphorylation by increasing transcriptional activation of PGC-1α , 14. SilA and B). To determine the specific location of NOX4 in the macrophage mitochondria, we isolated intermembrane space (IMS), inner membrane (IM), and matrix  (C). NOX4 was not present in the IMS or IM (D and E); however, NOX4 was localized in the matrix of macrophages exposed to asbestos over a prolonged time course (F and G).Asbestos increased NOX4 expression in mitochondria in a time-dependent fashion. , A and Blast, M) C. NOX4 wMS or IM , D and Ee course , F and GH and I). Similar observations were found when TIM22 was silenced (J and K). TIM23 is the translocase in the inner membrane that regulates the movement of proteins destined to mitochondrial matrix (L and M). TIM23 is only found in the mitochondria and is not found in the cytoplasmic fraction (N).Mitochondrial protein translocation is a regulated process driven by various mitochondrial translocases . The outsilenced , J and Kl matrix . Silencil matrix , L and Mfraction N.B and C). Overexpression of NOX4 had a similar increase in the localization.The port of entry for mitochondrial proteins to move into the mitochondria is facilitated by TOM complex that directs the protein transport to specific mitochondrial compartments . We founA and B). Overexpression of NOX4 increased TIM23 to a similar level as asbestos exposure. To visualize this interaction, cells were stained with NOX4 and TIM23 antibodies. Colocalization of NOX4 with TIM23 occurred in asbestos-exposed macrophages. Overexpression of NOX4 increased the positioning of both proteins together in the presence or absence of asbestos (C). Asbestos exposure (Pearson’s r = 0.849) and NOX4 overexpression (Pearson’s r = 0.884 and r = 0.897) was highly correlated with TIM23 (D and E).To determine if the colocalization of TIM23 and NOX4 was secondary to NOX4, we overexpressed NOX4. Asbestos increased both TIM23 and NOX4 , A and Basbestos C. Asbestth TIM23 , D and Ein vitro experiments. To test if these findings were present in vivo, we utilized mice harboring a conditional deletion of Nox4 in macrophages. Nox4fl/fl and Nox4−/−Lyz2-cre mice were exposed to man-made vitreous fiber (MMVF) or chrysotile asbestos (A). At day 21, the percentage of macrophages in the BAL were greater than 90% of the cells in all mice (D and E).We found that TIM23 is responsible for translocation of NOX4 protein to the mitochondrial matrix in macrophages in our asbestos A. At dayall mice , D and EA). Silencing NOX4 showed that asbestos did not increase TIM23 confirming that NOX4 is involved in transcriptional regulation of TIM23 (B and C).We questioned if NOX4 and TIM23 expression was regulated by transcription or secondary to protein degradation. We found that MG132 treatment does not alter the localization of NOX4 in mitochondria A. Silencof TIM23 , B and CNox4fl/fl mice compared to the mice exposed to MMVF (B and C). As expected, there was only TIM23 seen in the Nox4−/−Lyz2-cre mice. NOX4 was highly correlated with TIM23 in Nox4fl/fl mice exposed to asbestos in macrophages (Pearson’s r = 0.919) . This was not seen in the NOX4−/− mice. These observations validate that NOX4 and TIM23 colocalize in macrophage mitochondria from asbestos-exposed mice.To extend our findings in BAL macrophages, we questioned if there was localization of NOX4 and TIM23 in other macrophages in lung tissue. We found that NOX4 and TIM23 colocalized in lung macrophages in the /fl mice , F and GA), and isolated mitochondria (B and C) was subjected to NOX4 immunoprecipitation. Asbestos exposure increased TIM23 expression nearly 2-fold greater than control.We questioned if there was a direct interaction between NOX4 and TIM23. Immunoblot analysis of TIM23 was present in cells lysates A, and ischondria , B and CD and F). We confirmed that this interaction occurred in isolated mitochondria from asbestos-injured Nox4fl/fl mice, whereas it was not present in the macrophage mitochondria from Nox4−/−Lyz2-cre mice (E)Asbestos-induced injury significantly increased NOX4-TIM23 direct interaction compared to the mice that were exposed to MMVF , D and Fcre mice EG). There was direct interaction with the full-length GST-NOX4 and the GST-NOX4 447aa mutant, whereas TIM23 was absent with immunoprecipitation of all other constructs (H), suggesting that a putative motif from amino acids 213 to 447 are required for NOX4-TIM23 binding. In aggregate, these observations validate that NOX4-TIM23 direct interaction within the TMB region is required for NOX4 binding to TIM23 and the import into the mitochondria in lung macrophages from asbestos-exposed mice.NOX4 protein is composed of one transmembrane (TMB) domain, a FAD domain, and a NADPH domain. At the N terminus a 74 amino acid mitochondrial leader sequence (MLS) guides the protein to mitochondria, so we performed truncated mutations in the TMB domain instead of in the MLS. We cloned the GST-NOX4 mutant constructs that would correlate with amino acid residues at 124aa, 143aa, 213aa, 447aa, and NOX4 full-length mutation. The cloning construction was introduced at 372 nt, 429 nt, 639 nt, and 1341 nt, and NOX4 full-length protein is 1734 nt G. There nstructs H, suggesA and B). These observations are in support of our hypothesis that direct interaction of TIM23 and NOX4 is critical for the localization of NOX4 in mitochondria in humans subjects with asbestosis.We have shown that lung macrophages from asbestosis subjects have increased mitochondrial NOX4 in addition to increased mitochondrial ROS production , 11. To A–C) Because macrophages with profibrotic polarization have increased CPT1A and metabolic reprogramming to fatty acid oxidation (D–G) In contrast, Cpt1a and NOX4 were significantly reduced when TIM23 was silenced. Similar results were found in the nuclear and mitochondrial fractions of asbestos-exposed macrophages when NOX4 was silenced (H–J).Emerging data show a potential role of mitochondrial translocases in metabolic reprogramming , 18. We xidation , we nextxidation , D–G In silenced , H–J.FigK). OCR in lung macrophages from asbestos-exposed Nox4fl/fl mice was significantly increased, while Nox4−/−Lyz2-Cre mice exposed to MMVF or asbestos for 21 days were at the MMVF control level . Taken together, these observations strongly suggest that the direct interaction of NOX4 with TIM23 is necessary for metabolic reprogramming to oxidative phosphorylation in lung macrophages . Asbestos increased OCR in cells transfected with the control shRNA, whereas OCR was significantly reduced when TIM23 was silenced K. OCR inrophages .2.- and H2O2 production in macrophages (A and B).ROS from the mitochondria are known to have an important role in pulmonary fibrosis in multiple cells, including lung macrophages , 23, 24.rophages , A and BC). The mitochondrial to nuclear DNA ratio was significantly reduced in cells with NOX4 or TIM23 silenced (F). In cells transfected with the scrambled siRNA, asbestos markedly increased mDNA/nDNA.We asked what the impact of NOX4 and TIM23 interaction had on mitochondrial dynamics. We found that Drp1 was reduced when NOX4 or TIM23 was silenced, whereas asbestos significantly increased Drp1 expression in cells with the scrambled siRNA C. The misilenced F. In celD and E). Taken together, these data indicate that NOX4 interacts with TIM23 to enter the mitochondrial matrix, which is required for mitochondrial ROS production. NOX4 transcriptionally regulates TIM23, and this interaction regulates mitochondrial dynamics, specifically biogenesis.Expression of mitochondrial fusion proteins, Mitofusin1 and Mitofusin2, was significantly increased when NOX4 or TIM23 was silenced, whereas asbestos-exposed macrophages with the scrambled siRNA significantly reduced Mitofusin1 and Mitofusin2 , D and Evia activation of Akt-mTOR and NF-κB signaling pathways . All the experiments were performed as per NIH guidelines. escribed . Six to 2 asbestos was used to expose the cells in vitro, as previously described  cell lines were obtained from ATCC. Cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific) with the following supplements: 10% fetal bovine serum and penicillin/streptomycin. All experiments were performed with 0.5% serum supplement . 10 μg/cescribed .The pcDNA3.1-hNOX4, pcDNA3 C-terminal GST pBSU6-shTIM23/CMV-eGFP, and pBSU6-shScr/CMV-eGFP were purchased from Addgene. Plasmids were transfected with X-tremeGene nine Transfection Reagent (Roche# 06365809001), according to the manufacturer’s protocol. After 24 to 72 h, cells were subjected to expose to vehicle or asbestos followed by collection for downstream purposes. All siRNA were purchased from Integrated DNA Technologies. Cells were transfected using Dharma-FECT 4 (Dharmacon# T-2004) or DharmaFECT 2 (Dharmacon# T-2002), according to the manufacturer’s protocol. Eight hours following transfection, media were replaced, and cells were allowed to recover for 24 to 72 h.Full-length and truncated human Nox4 cDNA were PCR amplified from pcDNA3.1-hNOX4 vector using primer sequence; hNox4 full length, 1734 nt: AAGCTTGGTACCGAGCTCGGATCCGCCACCATGGCTGTGTCC and CTTATCATCATCATCAACGGATCCGCTGAAAGACTCTTTATTG; hNox4 372 nt: CTTATCATCATCATCAACGGATCCCAGATGGGCAGCCACATG, hNox4 429 nt: CTTATCATCATCATCAACGGATCCTGCATTCAGTTCAACAAAG; hNox4 1-639 nt: CTTATCATCATCATCAACGGATCCCAGCAGCCCTCCTGAAAC; hNox4 1-1341 nt: CTTATCATCATCATCAACGGATCCATCCAACAGGGTGTTGAG. The PCR products were cloned into BamH1 digested pcDNA3-c-terminal GST vector by Gibson Assembly kit  as company’s instruction. The correct full-length or truncated hNox4 sequences were confirmed by DNA sequencing.g for 10 min at 4 °C. The supernatant was collected and kept at 4 °C, while the pellet was lysed, homogenized, and centrifuged again in similar manner. The two supernatants were pooled together and centrifuged at 12,000g for 15 min at 4 °C. The pellet was the mitochondrial fraction, and supernatant was the cytoplasmic fraction. The pellet was washed twice in the mitochondrial buffer I and resuspended in mitochondria buffer II without sucrose. Mitochondria was used for mitochondrial protein analysis.Mitochondria and nuclei were isolated as previously described . Brieflyg for 10 min. The supernatant was collected as intermembrane space fraction, and the pellet was collected for further inner membrane isolation according to protocol , isolated mitochondria were incubated in a 1:5 volume of ice-cold hypotonic solution containing 10 mM Tris, pH 7.4, 1 mM EDTA, and 1 mM dithiothreitol and incubated for 10 min on ice. 150 mM NaCl was further added to the buffer for another 10 min on ice. Samples were centrifuged at 18,000g for 20 min at 4 °C, and the pellet was collected as mitochondrial matrix and dissolved in mitochondrial buffer II.Isolated mitochondria are subjected to isolation of inner mitochondrial compartments according to protocol , 48. Briprotocol . Pellet 2, 2 mM EDTA, and keep on ice for 15 min. Nonidet P-40 (10%) was added to lyse the cells, and the cells were centrifuged at 14,000 rpm at 4 °C. The nuclear pellet was resuspended in an extraction buffer containing 50 mm Hepes, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, and 10% glycerol for 20 min on ice followed by centrifuging at 14,000 rpm at 4 °C. The final supernatant was collected as nuclear extract.Nuclear isolation was performed by resuspending cells in a lysis buffer containing 10 mM Hepes, 10 mM KCl, 2 mM MgCl2-. and H2O2 production was determined fluorometrically as described previously  was performed as described . OCR meaImmunoprecipitation was performed as previously described . Equal aThe ratio of mitochondrial to nuclear DNA was determined as previously described .Total RNA was isolated from macrophages reverse transcribe, and quantitative real-time PCR was performed as described previously . Data wet test. The statistical analysis was expressed as ± SEM and p < 0.05 being significant. GraphPad Prism statistical software was used for all analysis.Statistical comparisons were performed using one-way ANOVA with a Tukey’s post hoc test or All the data in this manuscript are compiled and saved in “Carter Lab” on UAB server.This article contains The authors that they have no conflicts of interest with the contents of this article.


The development of biofuels, especially liquid hydrocarbon fuels, has been widely concerned due to the depletion of fossil resources. In order to obtain fuel precursors, the reaction of C–C bond formation is usually carried out with biomass derived ketones/aldehydes as reactants. Acetoin and 2,3-butanediol are two platform chemicals, which are co-existed in fermentation broth and traditionally separated by distillation, and then acetoin could be use as C4 building block to prepare hydrocarbon fuels. In order to mitigate the process complexity, direct aldol condensation reaction of acetoin in fermentation broth was studied in this work.10 fuel precursors and separation of C10 products and 2,3-butanediol from fermentation broth were achieved in one-pot with ethanolammonium butyrate (EOAB) and K2HPO4 as SOE reagents and catalysts. The SOE and reaction conditions such as the concentrations of EOAB and K2HPO4, reaction temperature and time were optimized. When the system was composed of 6 wt% EOAB-44 wt% K2HPO4 and the mixture was stirred for 6 h at 200 rpm, 40 ℃, the yield of C10 products was 80.7%, and 95.5% 2,3-butanediol was distributed to the top EOAB-rich phase. The exploration of reaction mechanism showed that an imine intermediate was rapidly formed and the subsequent C10 product formation was the key step for aldol condensation reaction.A one-pot process of product separation and acetoin derivative synthesis was proposed based on salting-out extraction (SOE). Aldol condensation reaction of acetoin and 5-methyl furfural in different SOE systems was compared, and the results showed that the synthesis of C2HPO4 as SOE reagents and catalysts, one-pot synthesis of fuel precursor from acetoin fermentation broth was achieved without prior purification. A yield of 80.7% for C10 products was obtained which was accumulated at the interface of two aqueous-phase, and 95.5% 2,3-BD was distributed to the top EOAB-rich phase. This work provides a new integration process of product separation and derivative synthesis from fermentation broth based on ionic liquid SOE.With EOAB and KThe online version contains supplementary material available at 10.1186/s13068-023-02344-w. Production of biofuels from renewable biomass is a sustainable and carbon-neutral way to replace fossil fuels gradually and reduce greenhouse gas emissions –3. A varAcetoin is one of promising sugar-derived platform chemicals , which cIn traditional way, pure compounds are used as raw materials to produce the derivative products , 4. Thus3PO4, and 92.7% of acetoin and 95.0% of 2,3-BD were recovered from the fermentation broth using the system of ethanolammonium butyrate (EOAB) and K3PO4 [Salting-out extraction (SOE) is a convenient and low-cost way for the primary separation of bio-based chemicals from fermentation broths . By saltnd K3PO4 . In addind K3PO4 . For exa10 fuel precursors which were accumulated in the middle phase, and 2,3-BD was partitioned into the top EOAB-rich phase, thus the derivative formation and product separation were achieved in one-pot.Encouraged by the application of EOAB in the SOE and EOAA in the aldol condensation reaction , 22, the2HPO4 was purchased from MacLean Reagent Co., Ltd. (China). Ethanolamine  and butyric acid  were purchased from Sinopharm Chemical Reagent Co., Ltd (China), and the ionic liquid EOAB was synthesized by the acid–base neutralization method [Acetoin , 2,3-BD  and 5-MF  were purchased from Aladdin Reagent Co., Ltd. (China). Anhydrous Kn method .Bacillus subtilis (CGMCC No. 23179), a mutant strain of B. subtilis DL01 which was isolated from sea sediment [4)2HPO4, 8 g/L KH2PO4, and 0.1 g/L MnSO4 (pH7.0), and the fermentation medium was composed of 140 g/L glucose, 7 g/L yeast extract, 12 g/L corn steep liquor powder, 6 g/L KH2PO4, and 0.13 g/L MnSO4. The fed-batch fermentation was carried out in a 5-L stirred bioreactor  with working volume of 2 L. The preserved strain was grown for 12 h at 37 ℃, 200 rpm in the LB medium, and then transferred to the seed medium  and cultured for 14 h at 37 ℃, 200 rpm. The obtained seed was inoculated  into the fermentation medium. The fermentation was carried out for 80 h at 37 ℃ under the oxygen supply condition of 0.25 vvm, 350 rpm. Glucose and (NH4)2HPO4 (mass ratio = 10:1) was supplemented when residual glucose in fermentation broth was less than 20 g/L. During the fermentation process, the pH value was not adjusted by automatic addition of 5 mol/L NaOH unless the pH value was less than 5.7. The fermentation broth was centrifuged at 5000 rpm for 30 min. The supernatant was stored at – 20 ℃ for the following SOE and reaction experiments. The concentrations of components in the supernatant were as follows: acetoin 65 g/L, 2,3-BD 40 g/L, citric acid 10 g/L and lactic acid 5 g/L.The fermentation broth was prepared by fed-batch fermentation with industrial glucose as a carbon source. sediment , was useAn aqueous solution containing 65 g/L acetoin and 40 g/L 2,3-BD was prepared for the following experiment, which was also used as the control to check the effect of the impurities in fermentation broth on aldol condensation reaction.10 fuel precursors was accumulated in the middle phase, and the salt was rich in the bottom phase. The top phase was taken out for acetoin and 2,3-BD determination, and then ethyl acetate was added to extract C10 product and residual 5-MF for GC analysis. The conversion of 5-MF (αMF), and the selectivity (SC10) and yield (YC10) of C10 products were defined as follows:The reaction was carried out for 18 h at 37 ℃ and 200 rpm in a shaking incubator. Aqueous solution of acetoin and 2,3-BD 10 mL, 0.04 mol salt, and 1.1 g EOAB were added into a 50-mL conical flask and vortexed for dissolution. Then, 5-MF was added to an equal mole of acetoin for reaction. After reaction, the system was divided into three phases, in which EOAB and 2,3-BD were enriched in the top phase, C2HPO4 and EOAB on the separation of acetoin and 2,3-BD from fermentation broth was investigated. The fermentation broth and K2HPO4 were added into a 25-mL tube and vortexed until the salt dissolved, and then EOAB was added and vortexed for 1 min. The total weight of the system was 10 g, and the final concentration of K2HPO4 in the system was 35 wt%, 41 wt%, and 47 wt% while the final concentration of EOAB was 3 wt%, 6 wt%, and 8 wt%, respectively. The mixture stood at room temperature for 2 h. The concentrations of acetoin, 2,3-BD and EOAB in the top phase were analyzed. The partition coefficient (K) and recovery (Y) were defined as follows:Vt and V0 represented the volume of top phase and fermentation broth added, respectively; Cjt, Cjb and Cj0 represented the concentration of chemical j in the top phase, bottom phase, and fermentation broth, respectively.To obtain an appropriate SOE condition for the coupled aldol condensation reaction, the effect of different concentrations of K2HPO4 were added into a 50-mL conical flask and stirred for dissolution. Then, EOAB and 5-MF were added. The total weight of fermentation broth, K2HPO4 and EOAB was 20 g. The mole ratio of 5-MF to acetoin was 1:1.The reaction was carried out in a magnetic stirring water bath. The temperature and stirring speed varied from 40 ℃ to 60 ℃, and 200 rpm to 600 rpm, respectively. The fermentation broth and KThe concentrations of fuel precursors, 5-MF, acetoin, and 2,3-BD were analyzed by gas chromatograph  equipped with a capillary column and FID detector. The concentrations of acetoin and 2,3-BD were analyzed by a chiral capillary column BGB-174 (30 m × 0.25 mm × 0.25 μm), while 5-MF and fuel precursors were analyzed by a capillary column HP-5MS (30 m × 0.25 mm × 0.25 μm).The concentrations of EOAB, organic acids, and residual glucose in the fermentation broth were analyzed by HPLC equipped with an Aminex HPX-87H column (300 × 7.8 mm) and a refractive index detector (Waters 2414), and 5 mmol/L sulfuric acid was used as mobile phase at a flow rate of 0.6 mL/min.Each experiment was carried out in triplicate. The mean experimental values with standard deviations are given in the tables and figures.3PO4), while phase split was not observed with saturated aqueous solution of dipotassium hydrogenphosphate (K2HPO4), sodium carbonate (Na2CO3), ammonium sulfate ((NH4)2SO4), and sodium acetate (CH3COONa) at 25 ℃ due to the high hydrophilicity of ILs [2CO3) could catalyze aldol condensation reaction [3PO4, K2HPO4 and K2CO3 were effective catalysts for nucleophilic addition reaction [3PO4, K2HPO4, K2CO3, potassium pyrophosphate (K4P2O7), and potassium acetate (CH3COOK)) were selected for SOE and reaction, since alkaline salts could remove the α-H of the carbonyl group to form a carbon anion in nucleophilic addition reaction.Our previous work showed that hydroxylammonium ionic liquids could form aqueous two-phase with tripotassium orthophosphate  was about 99% when only 10% EOAB was used as catalyst, while the conversion of 5-MF (αMF) was only 23%. When only the salt K3PO4 or K2CO3 was used as catalysts, αMF was greater than 70% while SC10 was less than 60%, probably due to the steric effect of reactants. For example, in the reaction of formaldehyde and propionaldehyde catalyzed by K2CO3, a yield of 67.8% was obtained [4P2O7 or K2HPO4 was used as catalyst, SC10 was greater than 90%, while αMF was less than 30%, resulting in a low yield of C10 products (YC10).Different catalytic efficiency was observed in the reaction of acetoin and 5-MF when salt was existed. As shown in Table obtained . When K4αMF was significantly increased, while SC10 was decreased, except CH3COOK where both αMF and SC10 were increased. The SC10 obtained from the systems of EOAB–K3PO4 and EOAB–K2CO3 decreased to less than 10%, and the main product became an imine intermediate, which was identified in the later experiment, thus the yield went down greatly. Among these systems, the combination of K2HPO4 and EOAB demonstrated high αMF and medium SC10, so the highest YC10 of 66% was obtained under present condition. The concentration of K2HPO4 in this reaction was near to saturation, so it was possible to improve SC10 by reducing the amount of K2HPO4 in the system.The synergistic effect of catalysis could be observed when both salt and EOAB were existed. The value of 2HPO4. As shown in Fig. 2HPO4. Under the same condition, the partition coefficient and recovery of 2,3-BD were lower than that of acetoin, respectively, which was related to their structure and the extraction mechanism of EOAB [2HPO4 concentration was greater than 41 wt%, the 2,3-BD recovery was higher than 90% while acetoin recovery was higher than 95%. Under the condition of 8 wt% EOAB-47 wt% K2HPO4, the highest partition coefficients of acetoin and 2,3-BD were obtained, which were 221.8 and 59.2, respectively.The SOE of acetoin and 2,3-BD from fermentation broth was explored at different concentrations of EOAB and K of EOAB . The sal2HPO4 not only acted as extractant and salting-out reagent but also catalysts, so the effect of their concentrations on the aldol condensation reaction was also determined. Generally, the composition of fermentation broth is very complex, including organic acids, residual sugars and proteins, which might affect the catalytic efficiency. Therefore, pure chemicals were used in the derivatization of bio-based chemicals to prepare fuels while few studies were reported to use fermentation broth directly [ SC10, while αMF from the fermentation broth was a little lower than that from the aqueous solution, thus a slightly decreased YC10 was obtained. This result indicated that it was feasible to use fermentation broth for aldol condensation reaction without prior purification.In this work, EOAB and Kdirectly , 34, 35.2HPO4 on the aldol condensation reaction are shown in Table 2HPO4 concentration of 47 wt%, significant improvement of αMF was observed when EOAB concentration was increased from 3 wt% to 6 wt%, while SC10 decreased with increased EOAB concentration from 3 wt% to 10 wt%. Under the condition of 6 wt% EOAB, both αMF and SC10 changed little with the increasing concentration of K2HPO4. These results showed that high concentration of EOAB and K2HPO4 was not beneficial for the SC10 improvement, resulting in the increase of by-products.The influences of the concentrations of EOAB and KYC10 of 76.0% was obtained in range of 41 ~ 44 wt% K2HPO4. However, 2,3-BD recovery at 44 wt% K2HPO4 was 95.5%, higher than that at 41 wt% K2HPO4. Therefore, the system 6 wt% EOAB–44 wt% K2HPO4 was finally selected for the integration of reaction and 2,3-BD separation from the fermentation broth.Under the condition of 6 wt% EOAB, the highest αMF was improved with increase in temperature. The detection of residual 5-MF showed that at 60 ℃ αMF reached the highest of 97.7% in 4 h, while at 40 ℃ and 50 ℃ the highest αMF was obtained in 8 h. SC10 decreased linearly with time extension at 60 ℃. At 40 ℃ and 50 ℃, SC10 firstly increased and then decreased with time extension. The highest SC10 and YC10 were obtained at 40 ℃ after 6 h reaction.Generally, the aldol condensation reaction under alkaline condition is mild. For example, the reaction catalyzed by amino acids and other organic small molecules was performed at room temperature , 37, whi10 products was influenced by temperature. With temperature increasing, the selectivity of product I, III and IV decreased while the selectivity of product II increased. Typically, the selectivity of product IV decreased 29.4% when temperature increased from 40 ℃ to 60 ℃. Thus, higher temperature made the C10 products react further and more by-products were generated.As shown in Fig. YC10 at 40 ℃ and 45 ℃ was significantly higher than that at 50 ℃ and 60 ℃, and there was no significant difference between the YC10 at 40 ℃ and 45 ℃. Therefore, the influence of stirring speed on the reaction was explored under these two temperatures. Theoretically, high stirring speed could promote the mixing of substances in the system and facilitate the reaction because 5-MF is insoluble in water. In fact, there was no significant difference in αMF, SC10 and YC10 in the range of 200 rpm–600 rpm, indicating that the stirring speed of 200 rpm could provide sufficient mixing efficiency for the reaction. Therefore, the reaction condition of 40 ℃ and 200 rpm for 6 h was selected, and the yield of C10 products was 80.7%. Compared with the yield of 87% using pure acetoin and EOAA catalyst [To avoid the deviation of results caused by uneven sampling and long sampling intervals, the points with high yield at different temperatures Fig. C were recatalyst , the yie2HPO4, the time courses of 5-MF consumption and formation of products at 40 ℃ were monitored. As shown in Fig. 10 products increased slowly, indicating a slow formation rate compared with 5-MF consumption. Similar phenomenon has been reported in the study of formaldehyde and propionaldehyde catalyzed by hydroxylammonium ionic liquid [To explore the mechanism of co-catalysis of EOAB and Kc liquid .Fig. 5Ti1H NMR ; B product IIpentan-3-one); C product IIIpent-4-ene-2,3-dione); D product IVpent-1-en-3-one); E intermediatemethylene)amino)ethan-1-ol). Fig. S31H NMR spectra of product IV synthesized from 5-MF and AC. Fig. S41H NMR spectra of intermediate.


We report the first example of a synthetic iron complex that functionally mimics cytochrome P450 in 100% water using H2O2 as the oxidant. This iron complex, in which one methyl group is replaced with a phenyl group in either wing of the macrocycle, oxidized unactivated C–H bonds in small organic molecules with very high selectivity in water (pH 8.5). Several substrates (34 examples) that contained arenes, heteroaromatics, and polar functional groups were oxidized with predictable selectivity and stereoretention with moderate to high yields (50–90%), low catalyst loadings (1–4 mol%) and a small excess of H2O2 (2–3 equiv.) in water. Mechanistic studies indicated the oxoiron(v) to be the active intermediate in water and displayed unprecedented selectivity towards 3° C–H bonds. Under single-turnover conditions, the reactivity of this oxoiron(v) intermediate in water was found to be around 300 fold higher than that in CH3CN, thus implying the role water plays in enzymatic systems.Cytochrome P450, one of nature's oxidative workhorses, catalyzes the oxidation of C–H bonds in complex biological settings. Extensive research has been conducted over the past five decades to develop a fully functional mimic that activates O via oxoiron(v) formation, catalyzes highly efficient and regioselective oxidation of C–H bonds in water using hydrogen peroxide, and thereby acts as a functional model of cytochrome P450.Development of a molecular iron complex, which  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bonds, under mild physiological conditions using molecular oxygen (O2) or hydrogen peroxide (H2O2) as the terminal oxidant.1 The quest for synthesizing biologically or industrially significant molecules via C–H bond functionalization has driven researchers worldwide to develop synthetic model complexes of these oxygenases and other enzymes,2 which can also prove essential in deciphering the mechanism of action of natural enzymes. Synthetic models2 of heme enzymes (or Cyt P450), which are excellent structural models due to their ability to activate H2O2 to form the oxoiron(iv) porphyrin π–cation radical,2 showed limited ability in catalytic hydroxylation and were not scalable. In addition, they mostly function in organic solvents.Nature utilizes earth-abundant iron-based enzymes, such as cytochrome P450, for the highly selective oxidation of C–H and C2O2 as the oxidant and an acid source. Starting from less bulky and less selective Fe-TPA3 and Fe-MEP complexes3 to pyrrolidine containing pyridine-N4 ligand-based Fe-PDP,3 Fe-CF3PDP3 and related sterically bulky ligated catalysts ,4 they performed highly regioselective oxidation of C–H bonds in a scalable manner. Meanwhile, our group has developed a robust iron complex based on a biuret-modified tetra-amido macrocyclic ligand (Fe-bTAML)5 which, unlike Fe-N4 systems, can perform selective hydroxylation of tertiary C–H bonds containing arenes and heteroarene compounds using mCPBA as the terminal oxidant.5 It is worth mentioning here that none of the synthetically useful biomimetic non-heme model complexes utilizes the green solvent “water” as the predominant solvent under non-acidic conditions . Reports on the oxidation of C–H or CC bonds in water are quite rare and limited to small substrate scopes, poor yields, and poor selectivities. These include the use of expensive second row transition metals6 like ruthenium or rhodium, as well as earth-abundant metals7 like iron and manganese as oxidation catalysts. On the other hand, oxidation of methane to methanol using H2O2 in water has been achieved by iron based catalysts8 such as the μ-nitrido diironphthalocyanine complex8 and N-heterocyclic carbene-ligated iron(ii) complex.8 In 2020, a new heterogenous construct involving an immobilized iron porphyrin complex on a self-assembled monolayer on Au electrodes was implemented for the oxidation of organic substrates in an aqueous solution using H2O2.8 In summary, iron complexes that utilize H2O2 as an oxidant in 100% water under homogeneous conditions to catalyze selective and scalable oxidation of unactivated C–H bonds are unknown  in highly basic pH respectively, which were unable to oxidize strong sp3 C–H bonds. The fifth-generation Fe-bTAMLs reported so far also form non-catalytic μ-FeIV-oxo dimers in pH < 10 in the presence of H2O2. However, in CH3CN and CH3CN : H2O mixtures, Fe-bTAML activates mCPBA or NaOCl to generate the oxoiron(v) intermediate which is capable of oxidation of strong C–H bonds , both under single-turnover and catalytic conditions.5 However, no reaction occurred in 100% water.Fe-TAMLs are historically known to activate Hv) species, except in one case,10 cannot be generated in 100% water or 100% aq. buffer solutions chemically, photochemically, or electrochemically (the one reported is not reactive toward C–H bonds). Generation of oxoiron(v) is critical for catalytic hydroxylation reactions. Hence activation of H2O2 to catalytically hydroxylate C–H bonds via the formation of an oxoiron(v) intermediate has been an unrealized dream. Such systems would be the first functional models of the Cyt P450 that hydroxylates C–H bonds using H2O2 as the terminal oxidant.In TAMLs and bTAMLs reported so far, high-valent reactive oxoiron(v) intermediate in 100% water that is competent to oxidize strong C–H bonds. The reactivity of this intermediate was a few hundred fold higher than the reactivity in CH3CN. We also show, for the first time, that this newly modified bTAML can activate hydrogen peroxide in water and oxidize a wide variety of substrates containing 3° C–H bonds  and substrates containing CC bonds in a selective and scalable manner. Notably, the selectivity and reactivity in water far exceed those observed in CH3CN – which helps us understand how protic solvents like water are critical to such reactivity.In this paper, we report a new modified Fe-bTAML complex that can generate an oxoiron(4N)2[Fe-(Cl)], 2, where a phenyl group replaces a methyl group on either side of the biuret moiety (4N)2[Fe(Cl)] (2) was synthesized following a new methodology (9 steps) wherein (dl)-2-phenylpropanoic acid was chosen as the starting material. Before metalation, the macrocyclic ligand was crystallized , and crystallizes in an orthorhombic crystal system ) -bTAML (1A).5 The electrochemical properties of this complex in CH3CN revealed two reversible peaks with peak potentials at E1/2 = 0.227 V (ΔEp = 64 mV) and E1/2 = 0.786 V (ΔEp = 71 mV) vs. Ag/AgNO3 (0.01 M)  indicated that both involve one electron redox process, which may be assigned to the FeIV/FeIII and FeV/FeIV couples. In 100% water the E1/2[FeIV/FeIII] value has increased up to 0.63 V , albeit with a loss of reversibility ) . The com Fig. S24, similar Fig. S27. The pea Fig. S28. This mameta-chloroperbenzoic acid (mCPBA) or aqueous sodium hypochlorite (NaOCl) led to the formation of oxoiron(v) species (3) whose UV spectral features  are reminiscent of 1A′  (v)-TAML12 and 1A′5 respectively. The Mössbauer spectrum of 57Fe enriched 3 at 77 K showed characteristic δiso and ΔEq of −0.58 cm−1 and 4.11 cm−1, respectively  species (>60%). In addition to this, there was another species with δiso and ΔEq of −0.2 cm−1 and 3 cm−1, respectively, which may correspond to a monomeric FeIV species (23%) forming under the same reaction conditions. These values are nearly identical to that of oxoiron(v) species of bTAML, 1A′.5Two-electron oxidation of 2 in acetonitrile by either t of 1A′ . 3 was wt of 1A′ , HRMS   and S25†v) intermediate of Fe-bTAML (1A′) in CH3CN medium upon the addition of mCPBA or NaOCl occurs through the formation of the dimeric intermediate, μ-oxo-diiron(iv), [{(bTAML)FeIV}2-μ-oxo]2−.5 In fact, the addition of 0.5 equiv. of mCPBA to 1 led to the formation of the diamagnetic μ-oxo-diiron(iv)dimer via comproportionation of 1A and 1A′ (mCPBA converts the μ-oxo-diiron(iv)dimer to the oxoiron(v) 1A′ intermediate. However, unlike 1, complex 2 does not dimerize upon addition of 0.5 equiv. of mCPBA  and Fe(v) or formation of a new monomeric Fe(iv) species (S = 1). In summary, the installation of the phenyl groups did result in preventing dimer formation as was hypothesized. The oxoiron(v) species (3) was reactive towards the hydroxylation of unactivated C–H bonds and the mechanism observed was similar to that reported for oxoiron(v) FebTAML 1A′. However, 3 was found to be around 300 times more reactive toward C–H bonds in comparison to 1A′.Earlier studies have shown that the formation of the oxoiron intermediate in 100% water is extremely challenging. In 2016, Collins et al. reported a beheaded TAML activator that can stabilize oxoiron(v) species in pure water, although it was not shown to be reactive towards C–H or CC bonds.10 Barring this report, quantitative oxoiron(v) formation was not achieved using any synthetic model complexes including Fe-TAML and bTAML systems in 100% water.The formation and spectroscopic characterization of the oxoiron(mCPBA to the parent (Et4N)2[Fe(Cl)] complex 2 at 10 °C in >99.5% water (0.5% CH3CN) produced green-coloured species, whose UV-Vis showed spectral features at λmax of 442 nm and 630 nm that are similar to that obtained in CH3CN  species 3   , was ∼200 times faster  medium and also ∼2700 times faster than oxoiron(v) of regular bTAML (1A′). However, this low self-stability in water does not compromise its reactivity toward C–H bonds . Similar selectivity, conversion, and formation of the corresponding 3° hydroxylated product were also obtained for cis-/trans-dimethylcyclohexane and decahydronaphthalene. Especially remarkable is the 100% stereo- and regioselectivity (3°) observed for the trans-hydroxylated product during the oxidation of trans-isomers of dimethylcyclohexane and decahydronaphthalene. Earlier studies showed that oxidation of 3° C–H bonds in these substrates is difficult because 1,3-diaxial interaction and oxidation are preferred towards the 2° C–H bonds. For example, in oxidation using Fe-PDP complexes,3 the selectivity towards 2° C–H bonds over 3° C–H bonds in trans-dimethylcyclohexane is approximately 9-fold. Similarly, in the case of 1B catalyzed oxidation in CH3CN,5 24% and 62% of the 2° products were obtained for trans-isomers of dimethylcyclohexane and decahydronaphthalene respectively, along with 3° products. Based on these literature reports, the complete stereo retention observed in the hydroxylated product for these trans isomers with 2 in water is truly remarkable. For the determination of reaction rates in 100% water, 6-methylheptan-2-yl isonicotinate  and cyclohexanol13  were used as substrates due to their comparatively higher water-solubility and the presence of C–H bonds with BDEs >90 kcal mol−1 . Oxidation under single turnover conditions also led to the selective formation of 6-hydroxy-6-methylheptan-2-yl isonicotinate and cyclohexanone in 32 and 75% yield respectively . The second-order rate constants for 6-methylheptan-2-yl isonicotinate and cyclohexanol at 10 °C were determined to be 14.4  formed from regular FebTAML 1A) upon the addition of water in CH3CN.13 To further understand the role of water, the solvent kinetic isotope effect (SKIE) was estimated for cyclohexanol oxidation with D2O as the solvent maintaining pH using a buffered solution. An SKIE (k2OH/k2OD) value of 2 was observed in 100% D2O  in the form of hydrogen peroxide, which is also a 2ev) can only be formed via the addition of strong oxidants like mCPBA and NaOCl to 1.5 In contrast, the addition of hydrogen peroxide to 1 resulted in the formation of unreactive and non-catalytic μ-oxo-dimeric species   medium resulted in a green coloured species with the characteristic UV-Vis spectrum (λmax of 435 nm and 625 nm) (m/z 553.01165) ; reminiscent of 3  , HRMS (m3.01165)  and EPR  Fig. S53.3CN–H2O (phosphate buffer pH 8.5) showed the formation of both the oxoiron(v) species, with λmax at 620 nm, and a minor monomeric FeIV species, ” species initially formed decays with time while the concentration of oxoiron(v) increases with time continuously  in 100% water  in the presence of 10 mM cis-dimethylcyclohexane, exclusive formation of the 3° hydroxylated product was observed in high yield. This result encouraged us to perform catalytic reactions in 100% water.In 100% water medium, however, we were unable to fully form this intermediate under ambient conditions (4 to 27 °C), with hydrogen peroxide, as evident from both UV-Vis Fig. S54 and stop2O2 (2–3 equiv.), which was added using a syringe pump in a complete aqueous medium comprising pH 8.5 phosphate buffer , substrate , and He buffer . A wide cis- and trans-isomers of dimethylcyclohexane and decahydronaphthalene , –Boc and acetate protected adamantane derivatives  and papaveraldine  respectively. Besides, the benzylic position in the ibuprofen methyl ester (derivatized anti-inflammatory drug) was predominantly oxidized to the ketone (25) along with minor hydroxylation at the 3° C–H bond adjacent to the isopropyl group. In addition to this, cyclooctane, which contains only 2° C–H bonds, gets primarily oxidized to ketone (17) with an overall yield of 85% along with a minor hydroxylated product. Additionally, when cyclohexane was used as a substrate, cyclohexanol was predominantly formed  w.r.t. trans isomers (entries 2 and 4) is expected which can be attributed to the strain release in the transition state for the cis isomers, as was observed in our previous reports.3 Remarkably, in our case, despite having a statistically higher number of methylenic C–H bonds, there is no 2° C–H bond oxidation accompanied by 100% RC, unlike observed previously for 1B catalyzed C–H bond oxidation using mCPBA5 (where 98% RC was observed) and other previously reported non-heme complexes  for entries 2 and 4 was reported to be around 3 : 1 and 2 : 3 respectively.5 Thus, we believe that such regioselectivity with no secondary alcohol or ketone product has never been encountered before. Moreover, for substrate entries 10 and 27 (menthyl acetate), catalysis with 1B exhibited 25% and 15% 2° C–H bond oxidation , whereas catalysis with 2 showed none.Both omplexes  and S8†.via the formation of dimeric species, alkene epoxidation was successful albeit in CH3CN solvent with modest yields.13Apart from aliphatic C–H bond oxidation, epoxidation of alkenes was also performed . Full cov), and thereby selectively oxidized strong C–H bonds in water medium (pH 8.5 buffer). The oxoiron(v) intermediate generated in water was around 300 times more reactive towards C–H bonds than in CH3CN under single turnover conditions. Catalytic oxidation was performed on substrates containing C–H and CC bonds using H2O2 as the terminal oxidant in water. Several substrates (34 examples) that contained arenes, heteroaromatics, and polar functional groups were oxidized with predictable selectivity and stereoretention using steric, electronic, and stereoelectronic rules with moderate to high yields (50–90%), low catalyst loadings (1–4 mol%) and a small excess of H2O2 (2–3 equiv.). Mechanistic studies indicated the oxoiron(v) to be the active intermediate during the catalytic reactions and displayed unprecedented selectivity towards 3° C–H bonds. To summarize, for the first time, using H2O2 as the sole oxidant, an iron complex was synthesized that performs highly selective and efficient catalytic hydroxylation of strong C–H bonds and epoxidation of alkenes in 100% water. However, the exact nature of the oxoiron(v) in water is still not fully understood and requires further investigation.A new modified “picket fence” bTAML based iron complex was synthesized upon substituting one methyl group present in either wing of the macrocycle with one phenyl group. By doing this, we were able to fine-tune the reactivity of Fe-TAMLs from a peroxidase to a cytochrome P450 mimic. During oxidation reactions, this complex activated hydrogen peroxide to generate oxoiron(All the data supporting this article have been uploaded as part of the ESI.†2O2 along with kinetic studies with both H2O2 and mCPBA. C. D. performed stop flow UV experiments and Mössbauer analysis. A. D. and S. G. D. gave their inputs to improve the manuscript. S. J., P. D., and S. S. G. analyzed the data and wrote the manuscript. All authors have given approval to the final version of the manuscript.The concept of the work was designed by S. S. G. S. J. optimized and synthesized the molecular catalyst followed by its characterization. P. D. with the help of S. J. performed all the oxidation reactions and their optimizations with HThere are no conflicts to declare.SC-014-D3SC03495J-s001SC-014-D3SC03495J-s002


LN5P45, comprising a chloroacethydrazide moiety that covalentlyreacts to the active-site cysteine residue. LN5P45 showsoutstanding target engagement and proteome-wide selectivity in livingcells. Importantly, LN5P45 as well as other OTUB2 inhibitorsstrongly induce monoubiquitination of OTUB2 on lysine 31. We presenta route to future OTUB2-related therapeutics and have shown that theOTUB2 inhibitor developed in this study can help to uncover new aspectsof the related biology and open new questions regarding the understandingof OTUB2 regulation at the post-translational modification level.Ubiquitin thioesteraseOTUB2, a cysteine protease from the ovariantumor (OTU) deubiquitinase superfamily, is often overexpressed duringtumor progression and metastasis. Development of OTUB2 inhibitorsis therefore believed to be therapeutically important, yet potentand selective small-molecule inhibitors targeting OTUB2 are scarce.Here, we describe the development of an improved OTUB2 inhibitor,  Post-translational modificationof DUBs, including phosphorylation, ubiquitination, SUMOylation, andacetylation, can further fine-tune these processes.6Ubiquitination is an essential post-translationalprotein modificationin a plethora of cellular processes. Ubiquitin generally modifiestarget proteins through lysine residues, and occasionally on cysteine,serine and threonine residues, which is catalyzed by the sequentialaction of E1, E2, and E3 enzymes.7 Recent research progress revealed that elevatedOTUB2 expression correlates with tumorigenesis and metastasis.11 OTUB2 can promote tumorigenic progression of non-small cell lungcancer cells through deubiquitination of U2AF2, stimulation of theWarburg effect, and activation of AKT/mTOR signaling.10 Moreover, OTUB2 has been identified as a cancer stemnessand metastasis-promoting factor that deubiquitinates YAP/TAZ proteinsin breast cancer cells, whereby poly-SUMOylation on OTUB2 lysine 233mediates the interaction with YAP/TAZ.11 These studies demonstrate OTUB2 to be a relevant therapeutic target,and the development of OTUB2-specific inhibitors can potentially bebeneficial for cancer therapies.Ovarian tumor (OTU) domain-containing ubiquitin aldehyde-bindingprotein 2 (OTUB2) is an active-site cysteine containing DUB.14 there are hardly any reportson potent and selective inhibitorsof OTUB2. A previous high-throughput electrophilic-fragment screeningcampaign enabled identification of covalent OTUB2 inhibitor OTUB2-COV1,which showed in-cell activity and good selectivity for OTUB2 withinthe DUB family.15 However, the biologicalapplication of this compound is limited due to its moderate potency,and its improvement was restricted due to a lack of density of keyregions in the co-crystal structure of the OTUB2-inhibitor complexand unknown absolute stereochemistry of the inhibitor. Here, we reportthe development of improved OTUB2 inhibitors through optimizationof the original OTUB2-COV1 inhibitor with respect to its stereochemistryand aromatic ring substituents. For our best inhibitor, LN5P45, we solved the X-ray co-crystal structure in complex with OTUB2,and we conducted a streamlined cysteine activity-based protein profiling(SLC-ABPP), which revealed that LN5P45 selectively engagesendogenous OTUB2 over other ubiquitin machinery components. Interestingly,while utilizing LN5P45 in cells, we unexpectedly foundthat this OTUB2 inhibitor also has a unique secondary activity, themonoubiquitination of the inhibited OTUB2 on lysine 31. This suggeststhat OTUB2 inhibition may have more surprises, which can be furtherexplored with this inhibitor.Despite numerous publicationson potent DUB inhibitors,1) canDUB-selectively inhibit OTUB2 with moderate potency in living cells,thereby forming an irreversible covalent bond with the OTUB2 activesite cysteine (Cys51).15 To further optimizethe inhibitory properties of this compound, we first turned our attentionto elucidating the preferred stereochemistry of the substituents attachedto the cyclopropane, as the exact nature of the stereocenters in COV-1were unknown. COV-1 is synthesized in two or three steps from 2-phenylcyclopropane-1-carboxylate(11) or its methyl (12) or ethyl ester (13) derivatives, as shown in Scheme S1. The stereochemistry in the final product originates from the stereochemicalnature of the starting compound and does not change during the courseof the synthesis. We acquired three commercially available stereoisomersof 2-phenylcyclopropane-1-carboxylate (11): a racemicmixture of the trans-only isomers, a racemic mixtureof the cis-only isomers, and the chirally pure isomer from which compounds 2–4 were synthesized, respectively and negative  controls. The inhibition was plottedagainst the compound concentration from which the IC50 valueswere calculated . Only the compounds withthe trans configuration inhibited OTUB2. Virtuallyno inhibition was observed for the cis configurationcompounds 3 and 4, whereas the IC50 value for compound 2 is nearly identical to that ofcompound 1. Hence, we reasoned that compound 1 most likely has the trans configuration and thatthis is required for OTUB2 inhibition.Previously, we showedthat the chloroacethydrazide-containing molecule COV-1  startingcompounds were obtained via enantioselective cyclopropanationof styrene and α-ethyl diazoacetate (EDA) using engineered myoglobin(Mb) carbene transferases developed by the Fasan lab (Scheme S2).20 Both trans enantiomers were produced enzymatically in enantiopure form andfurther processed to obtain the corresponding chloroacethydrazidecompounds 5 and 6 , whichwere tested for OTUB2 inhibition. From the obtained IC50 values , it became apparent that, althoughboth stereoisomers inhibit OTUB2, compound 6 having the configuration is the mostpotent one with an IC50 value of 6.8 μM, which makesit about twice as potent compared to its enantiomer compound 5 and the initial COV-1 compound.Next, we examined whether one of the S,2S) configuration as thepreferred stereoisomer, we turned our attentionto the aromatic ring substituents to further improve the compoundpotency. Applying the same chemo-enzymatic strategy described above,a panel of aryl substituted trans  cyclopropane compounds 7–10 were generated having a methyl (7–9) or fluorine (10) substituent at differentpositions on the aromatic ring. The compounds were tested for theirOTUB2 inhibitory potency using the assay as described above . Introduction of a methyl group at the para position (compound 9) or a meta, para-difluoride (compound 10) hadlittle effect on IC50, but the ortho-methylgroup had a detrimental effect (compound 7). Interestingly,introducing a meta-methyl group (compound 8) increased the inhibitory potency, resulting in an IC50 of 2.3 μM. Since these compounds are all covalent, irreversibleinhibitors,15 the IC50 dependson the incubation time. To get a better estimate of their respectiveinhibitory capacity, independent of incubation time, we determinedthe kinact/KI values of compounds 5–10 by pre-mixingthe compounds with the substrate before adding OTUB2. The obtainedvalues corroborated well with the IC50 values , again confirming compound 8 to bethe most potent, which was renamed LN5P45 for furtherexperiments and to ease future reference. The in vitro selectivity of LN5P45 for OTUB2 was assessed usinga panel containing six DUBs from the OTU family, including its closestfamily members OTUB1, OTUD1, OTUD2, OTUD3, OTUD6B, and Cezanne, andtwo DUBs from outside this family, UCHL1 and USP16 . Most of these DUBs were not inhibitedby LN5P45 at concentrations up to 150 μM, and onlymarginal inhibition was observed for UCHL1 and USP16 with IC50 values above 100 μM. A slightly better potency was found forOTUD3 with an IC50 value of 56 μM, which demonstrates LN5P45 to be about 20-fold more potent toward OTUB2.After having determinedthe  for compounds analysis 1. Compar0 values 1. More iLN5P45 within the OTUB2 active site.LC–MS analysis of OTUB2 before and after reaction with LN5P45 confirmed the formation of a covalent complex by showinga mass difference of 230 Da, which corresponds to a substitution reactionat the chloride . To furtherobtain information on the structural aspects of the interactions,we solved the co-crystal structure of OTUB2 in complex with LN5P45 at 1.77 Å resolution .In contrast to the co-crystal structure of the OTUB2-COV-1 complex(PDB 5QIO),a clear density for the cyclopropyl-phenyl moiety in our OTUB2-LN5P45 co-crystal structure was detected, which is likelydue to the fact that LN5P45 has the defined  configuration. The phenyl ring moietypoints outside the protein toward the solvent and has as such no obviousinteractions . Its orientation, away from the protein surface, however couldexplain our earlier observation that only the compounds with the trans substituted cyclopropane ring (compounds 5–10) and not the cis-linkedones (compounds 3 and 4) inhibited OTUB2.The latter situation would likely result in a conformational clashof the phenyl group with the protein. This could also explain thefinding that the ortho-methyl-substituted compound 7 showed diminished inhibition. The trans orientation on the other hand nicely positions the inhibitor insidethe small groove leading to the active site. When comparing the co-crystalstructure to the initial compound COV-1 5QIO, both the phenyl moieties point outwardof the protein, while for LN5P45, we actually found theelectron density to validate this position . The binding of the acethydrazidepart with the OTUB2 active site compared well with earlier findingsfrom the PDB 5QIO structure (5QIO structure), Asn50,and Cys51. The carbonyl directly attached to the cyclopropane ringinteracts with the side chain of His224 through a hydrogen bond, andthe Glu174 side chain carboxylate also forms hydrogen bonds with bothof the hydrazide amines.We next set out to evaluate thebinding mode of chloride S3. To fuchloride S3. To fuchloride S3. To fuLN5P45 also can engage with endogenous OTUB2, we used thebone-metastatic (BM) derivative of breast cancer cell line MDA-MB-231,which was reported to express relatively high levels of OTUB2 comparedto other breast cancer lines.11 Becauseof the lack of a good antibody that selectively recognizes endogenousOTUB2, siRNA-mediated depletion of endogenous OTUB2 was performedas a control to confirm OTUB2 labeling with our Rho-Ub-PA probe, anddepletion of OTUB2 was confirmed by real time quantitative polymerasechain reaction (RT-qPCR). This enabled the identification of a specificprotein band (around 35 kDa) that was substantially reduced in theOTUB2-depleted sample . Theobserved molecular weight of this labeled protein matched with theexpected molecular weight of Rho-Ub-PA-labeled OTUB2 (OTUB2 27.2 kDa+ 8 kDa Ub probe). Moreover, upon incubation of living BM MDA-MB-231cells with 10 or 20 μM LN5P45 for 4 h, the Rho-Ub-PA-labeledOTUB2 band almost disappeared compared to the DMSO control sample. Since none of the other Rho-Ub-PA-labeledbands were affected by the OTUB2 inhibitor, this result indicatesthat LN5P45 can specifically inhibit endogenous OTUB2.Next, we endogenously tagged OTUB2 with GFP in HeLa cells using CRISPR-Cas9.23 Three independent GFP-positive clones  were validatedby OTUB2-specific siRNA treatment and subsequent anti-GFP immunoblottingand flow cytometry . As clone#9 cells mainly expressed a smaller non-specific GFP-containing protein,we only used clones #11 and #32 for subsequent studies. Like the transientlytransfected HEK293T cells .To validate that d sample S5. Theod sample S5. TheoLN5P45 up to 25 μMin these cells . To further investigatethe selectivity of LN5P45 throughout the proteome, weperformed SLC-ABPP to determine its cysteine targets.24 For this, we incubated HeLa cells that ectopically expressedeither GFP or GFP-OTUB2 WT with either DMSO or 10 μM LN5P45, followed by cell lysis and incubation with a desthiobiotin iodoacetamide(DBIA) probe that is used to distinguish reactive cysteine sites thatare not bound to LN5P45 (LN5P45 versus DMSO treated)for both the HeLa cells expressing GFP and GFP-OTUB2 . Of note, theslower-migrating OTUB2-inhibition-induced X band was independent ofthe presence of the GFP tag of GFP-OTUB2, as a similar slower migrating(X′) band was detected with a Flag antibody in cells ectopicallyexpressing Flag-HA tagged OTUB2 . These results support the hypothesis that OTUB2 inactivation incells induces OTUB2 modification.Interestingly, the Western blot analysisof GFP-OTUB2 in LN5P45-treatedHEK293T cells that expressed exogenous GFP-OTUB2 WT. Five proteinbands covering the X band area were excised .To elucidate the nature ofthe inhibitor-induced modification, we used GFP trap beads and SDS-PAGEto enrich the X band protein from lysates of  excised 4A and su excised 4B. This  excised 4C. Based excised 4C. Consi excised 4D. InterWe next set out to identify whichamino acid(s) is/are ubiquitinatedin GFP-OTUB2. Although peptide sequencing by mass spectrometry failedto locate the specific modification site, it helped to exclude severallysines. OTUB2 K12, K31, K37, K44, K46, and K221 were possible sites,and we therefore generated single lysine to arginine mutants of GFP-OTUB2for each of these sites. Analysis in HEK293T cells showed that onlyK31R mutation resulted in loss of the monoubiquitination band uponOTUB2 inhibitor treatment and that monoubiquitination of the catalyticinactive mutant C51S was not further increased by the inhibitor either4E.via lysine 31 and that inactivation of OTUB2 by catalyticsite mutation or inhibitor treatment leads to elevated monoubiquitination.This shows how inhibition of OTUB2 stimulates its modification, whichmay result in secondary effects in cells following OTUB2 inhibition.Altogether these data demonstrate that OTUB2 is ubiquitinated 25 In this study, we developed the improvedcovalent OTUB2 inhibitor LN5P45 through optimizationof the original OTUB2-COV1 inhibitor by addressing absolute stereochemistryand aromatic ring substituents. We determined the preferred absolutestereochemistry as , andthe LN5P45-OTUB2 co-crystal structure explained the moleculardetails of the inhibitor binding mode. The co-structure also revealedthat the aromatic moiety points outside the protein. For future inhibitoroptimizations, it could be worthwhile to introduce more hydrophilicresidues at the phenyl ring to accommodate for its solvent-exposednature. Our substituent analysis revealed that the meta position on the aromatic ring is the best site to introduce modifications.This opens the way to install fluorescent/affinity tags or E3 ligaseligands to create OTUB2-specific activity-based probes or PROTACS,respectively. As such, compound LN5P45 could aid in thedevelopment of future therapeutics that target diseases associatedwith elevated levels and/or increased activity of OTUB2.The inhibition of DUB activity is subject toa growing interestin drug development, and several successful examples of potent DUBinhibitors have been published in recent years.6 Monoubiquitinationof OTUB1, primarily at lysines 59 and 109, is critical for OTUB1 functionto suppress UbcH5 and induce p53,26 andthe DUB activity of Ataxin-3 and JOSD1 can be enhanced through ubiquitinationof lysine 117 on Ataxin-328 and lysine 136 on JOSD1.29 As opposed to activation, the enzymatic activityof UCHL1 is restricted by monoubiquitination of lysine residues nearthe active site.30 In addition to theseactivity alterations, the subcellular localization of BAP1 is regulatedby multi-monoubiquitination within its NLS region.31 USP30 is ubiquitinated by Parkin for proteasome degradation.32 USP4 was shown to self-deubiquitinate afterbeing ubiquitinated by E3 ligase Ro52, and it was suggested that USP4forms a heterodimeric protein complex with Ro52 in which both proteinstransregulate each other.33 In addition,auto-deubiquitination of USP4 has been shown to stimulate homologousrecombination and its interaction with CtIP to recruit CtIP to DNAdamage sites, suggesting ubiquitination-dependent regulation of DUBenzyme interaction and function.34 However,we observed and report here for the first time the monoubiquitinationof a DUB in cells as a result of inhibitor treatment. Whether cellsput this mechanism into place to counteract the effect of inhibitionand whether it is unique to OTUB2 and/or this particular type of inhibitor remain to be investigated but may deliver valuable informationfor future drug development campaigns. Future research may unveilthe potential roles of OTUB2 monoubiquitination in the regulationof its enzymatic activity, localization, degradation, and other functionsand could lead to the identification of a similar effect for otherDUBs and inhibitors. The selective OTUB2 inhibitor identified andcharacterized in this study will be a critical lead for such studies.Interestingly,OTUB2 is monoubiquitinated upon inhibitor treatmentin cells and we were able to pinpoint Lys31 as the ubiquitinationsite. Ubiquitination of DUBs is a common post-translational modificationthat can regulate DUB function, localization, or stability, and severalexamples have been reported.Supporting Information.Synthetic procedures and characterizationdata for the OTUB2 inhibitorsare provided in the Table S1. The assays were performed in “nonbindingsurface flat bottom low flange” black 384-well plates (Corning3820) at room temperature in a buffer containing 50 mM Tris·HCl,100 mM NaCl, pH 7.6, 2.0 mM cysteine, 1 mg/mL 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonicacid (CHAPS), and 0.5 mg/mL γ-globulins from bovine blood (BGG).All dispensing steps involving buffered solutions were performed ona Biotek MultiFlowFX dispenser. Each well had a final volume of 20.4μL. The compounds were dissolved in 10 mM and 1 mM DMSO stocksolutions, and appropriate volumes were transferred to the empty platesusing a Labcyte Echo acoustic dispenser to obtain a 12-point serialdilution. A DMSO back-fill was performed to obtain equal volumes ofDMSO (400 nL) in each well. N-Ethylmaleimide  was used as a positive control (100% inhibition) and DMSO asa negative control (0% inhibition). A 10 μL portion of bufferwas added, and the plate was vigorously shaken for 20 s. Next, 7 μLof the DUB was added to reach the following final concentrations:OTUB1, 50 nM; OTUB2, 25 nM; OTUD1, 10 nM; OTUD2, 50 nM; OTUD3, 5 nM;OTUD6B, 10 nM; Cezanne, 5 nM; UCHL1, 1 nM; USP16, 2 nM, followed byincubation for 120 min. A 3 μL portion of the substrate was added , and the increase in fluorescenceover time was recorded using a BMG Labtech PHERAstar plate reader. The initial enzyme velocitieswere calculated from the slopes, normalized to the positive and negativecontrols, and plotted against the inhibitor concentrations using thebuilt-in equation “[inhibitor] vs response – variableslope (four parameters), least-squares fit” with constraints“Bottom = 0” and “Top = 100” in GraphPadPrism 8 software to obtain the IC50 values.An overview of the used recombinant DUBs is provided in kinact/KI determinations, the order of OTUB2 and substrate additionwas reversed and no incubation time was used. All data fitting andcalculations were done using GraphPad Prism 8 software. The fluorescenceintensities were plotted against time (in seconds) after a baselinecorrection using the DMSO control for each inhibitor concentration.The data were fitted to the equation FI = (vi/kobs)[1 – ek(obs)t–]. The thus obtained kobs values were plotted against the concentrationinhibitor, fitted to a linear regression, and the kinact/KI value was determinedfrom the slope.In thecase of the LN5P45 stock solution in DMSO  and incubated for 30 min at room temperature.Samples were then diluted 3-fold with water and analyzed by mass spectrometryby injecting 1 μL into a Waters XEVO-G2 XS Q-TOF mass spectrometerequipped with an electrospray ion source in positive mode  with a resolution of R = 26,000. Sampleswere run using two mobile phases: (A) 0.1% formic acid in water and(B) 0.1% formic acid in CH3CN on a Waters Acquity UPLCprotein BEH C4 column .Gradient: 2–100% B. Data processing was performed using WatersMassLynx mass spectrometry software 4.1, and ion peaks were deconvolutedusing the built-in MaxEnt1 function.Samples of 1.4 μM OTUB2 in 70 μL ofbuffer containing50 mM Tris·HCl, 100 mM NaCl at pH 7.6, and 2.0 mM cysteine wereprepared. These samples were treated with 1 μL of DMSO or 1μL of a 10 mM LN5P45-OTUB2 complexwas generated by incubating 40 μMpurified OTUB2 with a five-fold excess of the inhibitor for 1 h at37 °C in GF buffer .The formation of the covalent complex was confirmed using LCMS (Xevo)before being applied to a Superdex75 16/60  gel filtrationcolumn equilibrated in GF buffer. The peak fractions containing thecomplex were concentrated to 24.5 mg/mL and flash frozen in liquidN2, awaiting crystallization.For crystallization, the covalent Sitting drops wereset up in a 1:1 ratio with the mother liquor, and crystals appearedafter several days in conditions with 0.1 M HEPES pH 8.0, 8–20%(v/v) isopropanol and 10–16% PEG 4000. Single crystals werefrozen in mother liquor and 30% ethylene glycol as a cryoprotectantand shipped to Diamond Light Source  for diffraction.36 usingthe DLS computing grid. The CCP4 program suite37 was used: first data reduction with Aimless,38 followed by molecular replacement using PHASER39 with OTUB2 from PDB entry 5QIO as a search model.Iterative cycles of refinement using REFMAC40 and model building with COOT41 were performedbefore placing the LN5P45 inhibitor in the remainingdensity. Restraints for the inhibitor were generated using the GRADEwebserver  and used in further refinement. Dataprocessing and refinement statistics are reported in Table S2, and the final model is deposited in the ProteinData Bank (PDB ID: 8CMS).Crystalswere diffracted at beamline I04-1 at Diamond, and the resultingdata sets were processed with DIALS42 (laboratory of Peter ten Dijke) were culturedunder standard conditions in Dulbecco’s modified Eagle’smedium (DMEM) (Gibco) supplemented with 8% FCS (Biowest) and 1% penicillin/streptomycinat 37 °C and 5% CO2. All cell lines were tested formycoplasma contamination using a MycoAlert Mycoplasma Detection Kit on a monthly basis.HEK293T (Cat# ATCC CRL-3216),HeLa (ATCC), and bone metastatic (BM) MDA-MB-231 cellsFor siRNA transfections,OTUB2 siRNA oligos were purchased from Dharmacon. The siOTUB2 poolconsists of four individual siRNA’s, including (siGENOME Cat#MQ-010983-01-0002), siOTUB2_P1 (Cat# D-010983-01), siOTUB2_P2 (Cat#D-010983-02), siOTUB2_P3 (Cat# D-010983-03), and siOTUB2_P4 (Cat#D-010983-04). Silencing was performed in BM MDA-MB-231 cells as follows:for a six-well plate format, 200 μL of siRNA (500 nM stock)was incubated with 4 μL of Dharmafect reagent 1 (Dharmacon)diluted in 196 μL of medium without supplements  with gentle shaking for 20 minat room temperature (RT). A total of 80,000 cells resuspended in 1.6mL of growth medium were added to transfection mixes to a total volumeof 2 mL per well and cultured for 3 days prior to further analysis.For DNA transfections, HEK293T cells were seeded to achieve 50–60%confluence the following day and transfected using PEI  as follows: 200 μL of DMEM mediumwithout supplements was mixed with DNA and PEI (1 mg/mL) with a ratioat 1:3 , incubated at RT for20 min, and added drop-wise to the cells for culturing for 24 h priorto further analysis. The reaction was scaled using the same componentratios as follows: 12-well plate–1 μg DNA, 6-well plate–3μg DNA, 6 cm dish–8 μg DNA.21 HA-Ub,43 Flag-HA-OTUB2WT ,44 and pOPINK-OTUB245 constructs have beenpreviously described.GFP-OTUB2 WT and C51S mutant,46 In brief, a PCR mixture containinga GFP-OTUB2 WT template, mutation primers, Pfu buffer, dNTPs, TurboPfu Polymerase (Agilent), and MilliQ water up to 50 μL reactionvolume was subjected to PCR amplification using the following program:95 °C for 2 min  × 18 cycles; 68 °C for 20 min; 4 °Cforever. PCR products were digested with 1 μL DpnI (Thermo FischerScientific) for 2 h at 37 °C to remove the methylated DNA templateand then transformed into competent DH5α. All mutated constructswere verified by sequencing. For primer sequences, refer to Table S3. All primers were purchased from IDT.For site-directed mutagenesis of OTUB2K12R, K31R, K37R, K44R, K46R, and K211R, the same protocol was usedas before.47 , mouse anti-GAPDH , or mouse anti-β-actin .The following antibodies were used for detectionof endogenous and overexpressed proteins by immunoblots: mouse anti-Ubiquitin, mouse anti-HA , rabbit anti-mGFP23 In brief, HeLa cells were co-transfected withOTUB2/pX330 and OTUB2/endoGFP plasmids. Transfected cells were single-cellsorted by flow cytometry and validated by OTUB2 siRNA knockdown, DUBprobe labeling, and immunoblots.Endogenoustagging of OTUB2 was performed as described previously.OTUB2/pX330 plasmid was generatedby cloning OTUB2 5′ end-targeting guide sequence (gtcactatggtcagtgatcg)into pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid. OTUB2/endoGFP plasmidwas created by modifying the GFP-C1 vector, where the OTUB2 left homologyarm (agggggaggcatcctatcaaatggaccctaacgcataggctgaaggcacgtgaagtcccacccactttctatgacgtcccgcgtcccggcttctgattgcccgcctgagacgtcaatcgcagggcgtgtgtcttgctgggacacagtggaggtctaacctttggtttgcggagcggtcgggtgtattctccgccgcccccacgccctcgaggtccccgccaccgaaccagcggcggagcccgcccgcgcctcccgcggcattcccgcaccggatcgctcctcgctggggcgggacctggcctggcggctctggtcactatg)was inserted using NdeI/NheI sites (replacing the CMV promoter) andthe right homology arm (gtcagtgatcgtgggggatcgcgaagggggagcgggcagggggcgcggtgggcggggtcgctgccggagcgggtgcacccgcgggacgggggtcggacgcgaggctcagcccccagctcgcccccgccgctttccgaccccctgaaatacggagtccggacggatactgagggccaagtcgcgcccccctgtaccccgtggatgtgcagctgaggaggtccagctcggccccgagcccccgcccccagcgtgtccgccccaggtggcccggggcgccgcctcgacggcgctgggtgggcgccctcgacggaagcagggacaggga)using EcoRI/BamHI sites.n = 3 independent experiments.The mRNA level change of endogenous OTUB2 was assessedby RT-qPCR.Total RNAs were extracted using the NucleoSpin RNA II kit (MACHEREY-NAGEL)following the manufacturer instructions. Total RNA (1 μg) wasreversely transcribed using RevertAid First Stand cDNA Synthesis Kits(Thermo Fisher) to synthesize cDNA, and real-time quantitative PCRexperiments were performed using SYBR Green (Promega) in a CFX connectReal-Time PCR detection system (Bio-Rad). All the values for targetgene expression were normalized to GAPDH. Primers used for RT-qPCRare as follows: OTUB2 forward primer TTCTTCGGGACCATCCTGAAA and reverseprimer CCAGGTAGGAATAGCCCAAGG, GAPDH Forward primer TGCACCACCAAC TGCTTAGCand reverse primer CTCATGACCACAGTCCATGC. All the experiments wererepeated in 15 In brief, cell pelletswere resuspended in HR buffer , pH 7.4) and lysed by sonication at 4 °C. Clarified cell lysate (40 μg) was labeledwith a Rhodamine-Ubiquitin-propargylamide probe  at 37 °C for 30 min. Reactions were stopped bythe addition of LDS (lithium dodecyl sulfate) sample buffer  containing 2.5% β-mercaptoethanol,followed by boiling for 7 min.The in-cellinhibition of OTUB2 was assessed by using a DUB probelabeling assay.Samples were resolved on precastBis-Tris NuPAGE Gels  using MOPSbuffer . For fluorescencescan, labeled enzymes were visualized by in-gel fluorescence usinga Typhoon FLA 9500 imaging system  . For immunoblotting,proteins were transferred to a nitrocellulose membrane  at 300 mA for 3 h. The membranes wereblocked in 5% milk  in 1× PBS, incubated with a primary antibody dilutedin 5% milk in 0.1% PBS-Tween 20 (PBST) overnight at cold room, washedthree times for 5 min in 0.1% PBST, incubated with the secondary antibodydiluted in 5% milk in 0.1% PBST for 1 h, and washed three times againin 0.1% PBST. The signal was visualized using a LICOR Odyssey system.The intensity of bands was quantified using Image Studio software.46 Harvested cell pellets were lysed in 300 μL of lysisbuffer 1 , and protease inhibitors ). Then, 100 μL of lysis buffer 2  was added to thecrude lysates; samples were sonicated ; and SDS was subsequentlydiluted by bringing the sample volume to 1 mL with lysis buffer 1.After centrifugation ,lysates were incubated with 6 μL of GFP Trap Agarose (Chromotek)overnight at 4 °C. Beads were washed five times with lysis buffer1 and denatured with sample buffer by heating at 95 °C for 7min. Samples were subjected to 4–12% SDS-PAGE, and gel sampleswere either sliced for protein mass spectrometry detection or transferredto Nitrocellulose membranes for immunoblots, as indicated.To check the PTM on GFP-OTUB2,GFP trap pulldown assays were applied as before.via a homemade analytical nano-HPLC column . The gradient was run from10% to 40% solvent B (20/80/0.1 water/acetonitrile/formic acid (FA)v/v) in 30 min. The nano-HPLC column was drawn to a tip of ∼5μm and acted as the electrospray needle of the MS source. TheLUMOS mass spectrometer was operated in data-dependent MS/MS modefor a cycle time of 3 s, with a HCD collision energy at 32 V, andthe MS2 spectrum was recorded in the orbitrap. In the master scan(MS1), the resolution was 120,000 and the scan range was 400–1500at an AGC target of 400,000 at a maximum fill time of 50 ms. Dynamicexclusion after n = 1 with an exclusion durationof 10 s. Charge states 2–5 were included. MS2 precursors wereisolated with the quadrupole with an isolation width of 1.2 Da. Thefirst mass was set to 110 Da. The MS2 scan resolution was 30,000 withan AGC target of 50,000 at a maximum fill time of 60 ms.For MS analysis,gel slices were subjected to reduction with dithiothreitol, alkylationwith iodoacetamide, and in-gel trypsin digestion using a ProteineerDP digestion robot (Bruker). Tryptic peptides were extracted fromthe gel slices, lyophilized, dissolved in 95/3/0.1 v/v/v water/acetonitrile/formicacid, and subsequently analyzed by on-line C18 nanoHPLC MS/MS witha system consisting of an Easy nLC 1000 gradient HPLC system  and a LUMOS mass spectrometer (Thermo). Samples wereinjected onto a homemade precolumn  and eluted 24 In short, HeLa cells, transfected with GFP orGFP-OTUB2 WT with PEI reagent for 24 h, were grown to 80% confluenceand incubated with either DMSO or 10 μM LN5P45 for4 h in complete medium. Cells were harvested, lysed by sonicationin ice-cold PBS, and centrifuged at 15,000 RPM for 2 min to removecell debris. Protein concentrations were then determined by a BCAGold protein assay. Proteomes were normalized to 1 mg/mL in 100 μLfor each sample.SLC-ABPPprofiling was performed as described before.LN5P45-treatedproteome was labeled with the 500 μM DBIA probe for 1 h in thedark at room temperature (RT). Excess DBIA, along with disulfide bonds,was quenched and reduced using 5 mM dithiothreitol for 30 min in thedark at RT. Reduced disulfide bonds were alkylated using 20 mM iodoacetamidefor 30 min in the dark at RT. Proteins were precipitated using chloroform/methanol,re-solubilized in 40 mM HEPES pH 8.4, and digested using TPCK-treatedtrypsin and endoLysC (1:12.5 enzyme/protein ratio) overnight at 37°C. Digested peptides were labeled using TMTpro16-plex reagentsin a 1:4 ratio by mass (peptides:TMT reagents) for 1 h at RT. ExcessTMT reagent was quenched with 5 μL of 6% hydroxylamine for 15min at RT. Next, samples were mixed 1:1 across all TMT channels, andthe pooled sample was dried using a Speedvac.Each DMSO- and 4HCO3 pH 8.4. Driedsamples were dissolved in 200 μL of 10 mM NH4HCO3 pH 8.4, loaded onto the column, washed 3× with 200 μLof 10 mM NH4HCO3 pH 8.4, and eluted into threefractions with 150 μL each of 10, 20, and 50% acetonitrile (in10 mM NH4HCO3). Samples were re-dried usinga Speedvac.Dried sampleswere reconstituted in 1 mL of PBS and enriched byPierce streptavidin magnetic beads  by rotating end-over-end overnight at 4 °C. Nonspecificbinding peptides were washed using the following procedure: 3 ×1 mL of PBS pH 7.4, 2 × 1 mL of PBS with 0.1% SDS pH 7.4, and3 × 1 mL of HPLC-grade water. DBIA probe-containing peptideswere eluted using 700 μL of 50% acetonitrile with 0.1% TFA,dried using a Speedvac, and desalted using a mini-SPE column . Thecolumn was washed with 200 μL of 90% acetonitrile and 3×200 μL of 10 mM NHvia a homemade analytical nano-HPLC column. The gradient was run from 2 to 40%solvent B (20/80/0.1 water/acetonitrile/formic acid (FA) v/v) in 120min. The nano-HPLC column was drawn to a tip of ∼10 μmand acted as the electrospray needle of the MS source. The mass spectrometerwas operated in data-dependent MS/MS mode for a cycle time of 3 s,with a HCD collision energy at 36 V, and the MS2 spectrum was recordedin the orbitrap, with a quadrupole isolation width of 1.2 Da. In themaster scan (MS1), the resolution was 120,000 and the scan range was350–1600 at a standard AGC target at a maximum fill time of50 ms. A lock mass correction on the background ion m/z = 445.12 was used. Precursors were dynamicallyexcluded after n = 1 with an exclusion duration of45 s and with a precursor range of 20 ppm. Charge states 2–5were included. For MS2, the first mass was set to 110 Da, and theMS2 scan resolution was 45,000 at an AGC target of 200% with a maximumfill time set to auto.Peptideswere lyophilized,dissolved in 0.1% formic acid, and subsequently analyzed by on-lineC18 nanoHPLC MS/MS with a system consisting of an Ultimate3000nanogradient HPLC system  and an Exploris480mass spectrometer (Thermo). Fractions were injected onto a cartridgeprecolumn and eluted Homo sapiens minimal database using Mascot v. 2.2.04 (www.matrixscience.com) forprotein identification. Mascot searches were done with 10 ppm and0.02 Da deviation for precursor and fragment mass, respectively, andthe enzyme trypsin was specified. Up to two missed cleavages wereallowed for gel slices and three missed cleavages for SLC-ABPP. Forgel slices, methionine oxidation and ubiquitination (GG) on lysinewere set as a variable modification; carbamidomethyl on Cys was setas a fixed modification. For SLC-ABPP, methionine oxidation, Acetyl(protein N-term) and DBIA on Cys were set as a variable modification.Carbamidomethyl on Cys and TMTpro on Lys and N-term was set as a fixedmodification. Peptides with an FDR < 1% were accepted. Abundanceratios were calculated by dividing the inhibitor-treated channelswith the DMSO-treated channels.In a post-analysisprocess, raw data were first converted to peak lists using ProteomeDiscoverer version 2.4 (Thermo Electron) and then submitted to theUniprot