STING is an innate immune sensor for immune surveillance of viral/bacterial infection and maintenance of an immune‐friendly microenvironment to prevent tumorigenesis. However, if and how STING exerts innate immunity‐independent function remains elusive. Here, the authors report that STING expression is increased in renal cell carcinoma (RCC) patients and governs tumor growth through non‐canonical innate immune signaling involving mitochondrial ROS maintenance and calcium homeostasis. Mitochondrial voltage‐dependent anion channel VDAC2 is identified as a new STING binding partner. STING depletion potentiates VDAC2/GRP75‐mediated MERC (mitochondria‐ER contact) formation to increase mitochondrial ROS/calcium levels, impairs mitochondria function, and suppresses mTORC1/S6K signaling leading to RCC growth retardation. STING interaction with VDAC2 occurs through STING‐C88/C91 palmitoylation and inhibiting STING palmitoyl‐transferases ZDHHCs by 2‐BP significantly impedes RCC cell growth alone or in combination with sorafenib. Together, these studies reveal an innate immunity‐independent function of STING in regulating mitochondrial function and growth in RCC, providing a rationale to target the STING/VDAC2 interaction in treating RCC. STING is a canonical innate immune sensor triggering type‐I interferon production. Here, an innate immunity‐independent STING function in governing renal cancer cell (RCC) proliferation is reported through regulating mitochondria calcium homeostasis by binding mitochondrial calcium transporter VDAC2, as well as its therapeutic potential in treating RCC. Viral and bacterial DNA, as well as damaged genomic and mitochondria DNA, are sensed by the cytosolic DNA sensor cGAS (cyclic GMP‐AMP synthase). Binding of DNA to cGAS activates its enzymatic activity by triggering cGAS dimerization and phase transition toward synthesizing a special unsymmetric cyclic dinucleotide 2′3′‐cGAMP. Subsequently, 2′3′‐cGAMP binds STING (stimulator of interferon response cGAMP interactr 1) on the endoplasmic reticulum (ER) to promote its trafficking to Golgi and other peri‐nuclear membrane‐coated compartments, where STING recruits TBK1 to phosphorylate IRF3. Phosphorylated IRF3 dimerizes and translocates into nuclei in triggering the expression of interferon β (IFNβ). This leads to expression of immune modulatory genes and activation of adaptive immunity. However, STING can also promote autophagy, lysosome‐mediated cell death of myeloid cells, and inhibition of viral replication via IFNβ ‐independent mechanisms. In addition, STING also plays a critical role in responding to RNA viruses including influenza A viruses (IAV), vesicular stomatitis virus (VSV), dengue virus (DENV) and others, through directly binding and modulating function of essential RNA sensors RIG‐I and MAVS and other mechanisms.Innate immunity serves as the first line of defense against infectious pathogens in vertebrates to restrain pathogen invasion and activate adaptive immunity. Pathogen‐associated molecular patterns (PAMPs) help distinguish between self‐ and non‐self‐materials and initiate recognition and activation of innate immunity.1 Nucl) and genetic animal models . Moreover, evidence from gt/gtSting (Sting knockout) mice suggests that activation of the STING signaling creates an immune‐responsive microenvironment that suppresses tumorigenesis, and STING loss has been observed in colon cancer, gastric cancer and melanoma and restrains IFN‐mediated tissue repair and T cell priming. Activating STING has been shown to improve therapeutic effects of immune‐checkpoint blockades. STING also exerts a protective effect in metabolic diseases such as alcoholic liver disease, cardiovascular diseases including myocardial infarction, aging, and others through promoting IRF3 phosphorylation and activation of innate immune signaling.Other than nucleotide sensing, hyperactivation of STING has been observed and connected with inflammatory diseases from both pathological perspectives  disease is caused by GOF mutations in STING18) anβ ‐independent T cell death by inducing ER stress in T cells. STING depletion has been shown to promote cancer cell growth via accelerated S‐phase entry and increased chromosome instability in the absence of an immune environment. Interestingly, STING activation can mediate mitochondrial damage that leads to renal fibrosis and renal failure. Although extensive studies demonstrate that STING is involved in controlling tumorigenesis and renal failure, there is no evidence of STING involvement in the development of RCC, especially through a non‐canonical and innate immunity‐independent manner.Innate immunity‐independent STING function has also been investigated but is less understood. To this end, although STING activation mediates T cell tumor infiltration, STING was also reported to mediate IFN T cells.29 STI22.1 Our analyses of the TCGA dataset identified gene amplification of STING  but not cGAS (MB21D1)  showed STING overexpression.33 Our) Figure . Further cGAS MB21  Figure . Depleti) Figure  led to r) Figure  and decr) Figure  with the) Figure . In cont) Figure  also red) Figure . We next2.2STING‐S366A knockin A498 cells by CRISPR  and mitochondrial ROS (by MitoSOX Red), but also mitochondrial calcium levels (by Rhod‐2 AM)  Figure  were incA Figure , suggest2.4 ATP2A2 (SERCA Ca‐ATPase located on ER membrane), and ATP1A1  . Binding of STING with VDAC2, ATP2A2, and ATP1A1 was confirmed in both A498  and mitochondrial calcium uniporters (such as VDACs). In this process, GRP75 (glucose‐regulated protein 75) at the mitochondria‐associated membrane (MAM) bridges IR3R interaction with VDACs. Given we observed STING depletion induced increased Ca2+ flux into mitochondria and upregulated mito‐ROS levels  and ECAR (extracellular acidification rate) by seahorse. STING‐depleted A498 cells displayed a decreased basal respiration rate compared with control cells and a decreased ATP‐driven synthesis ability Figure  may causβL, Raptor, and Sin1  could promote mTORC1/S6K activation in A498 cells  would be an approach to maximize tumor inhibition. In addition, considering that VDAC2 cysteine residue palmitoylation and oxidation were also previously detected by mass spectrometry, we analyzed if any of these cysteine mutations may affect VDAC2 binding to STING. We generated VDAC2‐C103A, C210A, C227A, and C103A/C210A/C227A (3A) mutants and found none of these mutants significantly affect VDAC2 binding to STING , we depleted the protein acyl transferases ZDHHC3 or ZDHHC7 using shRNAs Figure , mimicki65 2‐Bs Figure  and STINs Figure , 2‐BP trs Figure  to facils Figure . Notablys Figure  through s Figure  as STINGs Figure . Like STs Figure , 2‐BP trs Figure . As a res Figure , while ds Figure . Moreoves Figure . Interess Figure . Like STs Figure , 2‐BP tr2.8 and currently various targeted therapies have been approved in treating RCC patients in clinic including tyrosine kinase inhibitors , HIF inhibitors (belzutifan) and mTOR inhibitors (temsirolimus and everolimus), we next examined if 2‐BP exerts any synergistic effects with clinically approved agents. To this end, although 2‐BP reduced mTORC1/S6K activation in RCC cells  and THCA (thyroid carcinoma). If STING is also a vulnerability in these two types of cancers or if STING regulates mitochondria ROS/calcium homeostasis in STAD and THCA remains an interesting topic for further investigations.It remains unclear why unlike other cancers including colon cancer,21 gasTMEM173 (STING) gene is observed in different ethnic populations‐ for example, HAQ (R71H‐G230A‐R293Q) is dominant in East Asian, while Africans have no HAQ. If these various STING variants affect STING binding with VDAC2 and determine STING dependency for cell growth in cancers beyond RCC warrants further in‐depth investigations.Notably, the heterogeneity of e no HAQ.69 If 4l‐Glutathione (reduced) was purchased from Gold Biotechnology (G‐155‐25). 4‐hydroxy Nonenal (4‐HNE) (32100) was purchased from Cayman. Sulfobutylether‐β‐Cyclodextrin (HY‐17031), Sorafenib (HY‐10201A), and RSL3 (HY‐100218A) were purchased from MedChemExpress. Everolimus (A8169) was purchased from ApexBio Technology. Temsirolimus (T3574) was purchased from TCI. H‐151 (6675) was purchased from Tocris.Bafilomycin A1 (S1413), cycloheximide (S6611), erastin (S7242), MG132 (S2619), MLN4924 (S7109), S6K1‐I (S2163), tBHQ (S4990) and rapamycin (AY‐22989) were purchased from Selleck. 2‐bromohexadecanoic acid (2‐BP) (238422), puromycin (P8833), hygromycin (H3274), and blasticidin (15205) were purchased from Sigma. α antibody (5324), anti‐GRP75 antibody (3593), anti‐HA antibody (3724), anti‐HIF‐1α antibody (36169), anti‐IRF3 antibody (4302), anti‐MCU antibody (14997), anti‐p‐4E‐BP1 (Thr37/46) (2855), anti‐p62 antibody (88588), anti‐p70 S6 Kinase (2708), anti‐p‐Akt (Ser473) (4060), anti‐p‐eIF2α (Ser51) antibody (3398), anti‐p‐Histone H2A.X (Ser139) antibody (9718), anti‐p‐IRF3 (Ser396) antibody (29047), anti‐PLK1 antibody (4513), anti‐p‐p70 S6 Kinase (Thr389) (9234), anti‐p‐S6 Ribosomal Protein (Ser235/236) (4858), anti‐p‐S6 Ribosomal Protein (Ser240/244) (5364), anti‐p‐STING (Ser366) (50907), anti‐p‐TBK1/NAK (Ser172) (5483), anti‐TBK1 antibody (51872), anti‐Skp2 antibody (2562), anti‐STING antibody (for WB and IP) (13647), anti‐STING antibody (for IF) (90947), anti‐ubiquitin antibody (3936), anti‐VDAC2 antibody (9412), anti‐rabbit IgG, HRP‐linked antibody (7074) and anti‐mouse IgG, HRP‐linked antibody (7076) were obtained from Cell Signaling Technology. Anti‐cyclin A antibody (sc‐751), anti‐GAPDH antibody (sc‐47724), anti‐GST antibody (sc‐459), anti‐NQO1 antibody (sc‐32793), anti‐p27‐antibody (sc‐1641), anti‐PARP‐1‐antibody (sc‐8007), anti‐Ribosomal Protein S6 antibody (sc‐74459) and anti‐vinculin antibody (sc‐25336) were obtained from Santa Cruz Biotechnology. Polyclonal anti‐Flag antibody (F‐7425), monoclonal anti‐Flag antibody , anti‐α‐tubulin antibody (T‐5168), anti‐Flag agarose beads (A‐2220), anti‐HA agarose beads (A‐2095) and glutathione agarose beads (G4510) were obtained from Millipore Sigma. Anti‐ATP1A1 antibody (14418‐1‐AP), anti‐COX IV antibody (11242‐1‐AP), anti‐HIF‐2α antibody (26422‐1‐AP), anti‐Histone H2A.X antibody (10856‐1‐AP) and anti‐VDAC2 antibody (11663‐1‐AP) were obtained from Proteintech. Anti‐Nrf2 antibody (ab62352) was obtained from Abcam. Invitrogen Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (A11034), Invitrogen Alexa Fluor 594 goat anti‐mouse IgG (H+L) (A11032), and Invitrogen Alexa Fluor 594 Donkey anti‐Rabbit IgG (H+L) (A21207) were obtained from ThermoFisher Scientific.All antibodies were used at a 1:1000 dilution in TBST buffer with 5% bovine serum albumin or non‐fat milk for western blotting. Anti‐Akt antibody (4691), anti‐ATP2A2 antibody (9580), anti‐calnexin antibody (2679), anti‐cGAS antibody (83623), anti‐CHOP antibody (2895), anti‐c‐Myc antibody (18583), anti‐cyclin B1 antibody (4135), anti‐eIF2The renal cell carcinoma tumor specimens were obtained from the University of North Carolina at Chapel Hill Lineberger Comprehensive Cancer Center Tissue Procurement Facility reviewed and approved by the Office of Human Research Ethics as described previously.71−1 streptomycin unless otherwise stated.Human renal cell carcinoma cell lines 786‐o, A498, ACHN, Caki‐1, RCC4, RCC10, RXF‐393, UMRC2, and UMRC6, human immortalized kidney cell lines HEK293, HEK293T, and HKC‐8, human cervical adenocarcinoma cell line HeLa and human glioblastoma cell line U87MG were cultured in DMEM medium supplemented with 10% FBS. Human benign prostatic hyperplasia cell line BPH‐1 cells were cultured in RPMI‐1640 medium supplemented with 10% FBS. All cell culture media were supplemented with 100 units of penicillin and 100 mg mL Packaging of lentiviral shRNA or cDNA expressing viruses, as well as subsequent infection of various cell lines were performed according to the protocols described previously. Following viral infection, cells were maintained in the presence of hygromycin (200 µg mL−1) or puromycin (2 µg mL−1), depending on the viral vector used to infect cells. Gene knockdown in shRNA‐tet‐on cells was achieved by refreshing medium containing 1 µg ml−1 of doxycycline  every 3 days.Cell transfection was performed using lipofectamine 3000 or polyethylenimine (PEI), as described previously.71 PacSTING into pCDNA3.0 vector using primer sets listed below. Flag‐STING‐HAQ‐H232R was constructed by PCR cloning HAQ‐H232R variant from THP‐1 cell cDNA into pCDNA3.0 vector. Flag‐STING‐H232R was constructed by PCR cloning H232R variant from hORFeome V5.1 collection into pCDNA3.0 vector. Flag‐STING‐HAQ‐Y199C‐H232R was constructed by overlapping PCR to clone HAQ‐H232R variant into pCDNA3.0 vector using primers listed below. pBabe‐Flag‐STING‐WT, C91A and C88A/C91A were constructed by PCR cloning WT, C91A and C88A/C91A into pBabe‐hygro vector. pLenti‐Flag‐STING‐WT, C88A/C91A and R238A were constructed by cloning WT, R238A and C88A/C91A into pLenti‐hygro vector. CMV‐GST‐STING‐WT and S366A were constructed by (overlap) PCR cloning WT and S366A into CMV‐GST vector. Flag‐STING‐1‐110, 1–150, 42–150, 82–150, 111–150, and 151–379 were constructed by PCR cloning respective STING truncations into CMV‐GST vector using primer sets listed below. Flag‐cGAS was as described previously. His‐Ub was as described previously. HA‐VDAC2‐WT, C103A, C210A, C227A, C103A/C210A/C227A (3A), E84Q, Q101K, S128A, and L136F were constructed by (overlap) PCR cloning VDAC2 into pCDNA3 vector using primer sets listed below. pLenti‐HA‐VDAC2 and ATP2A2 were constructed by PCR cloning VDAC2 and ATP2A2 into pLenti‐GFP‐hygro vector using primer sets listed below. CMV‐GST‐GRP75 was constructed by PCR cloning GRP75 from hORFeome V5.1 collection into CMV‐GST vector. CMV‐GST‐ZDHHC7‐WT and C160S were constructed by (overlap) PCR cloning ZDHHC7 into CMV‐GST vector using primer sets listed below.Flag‐STING‐WT, C88A, C91A, C88A/C91A were constructed by (overlap) PCR cloning eviously.73 HisSTING‐BamHI‐F: GACACCGACTCTAGAGGATCCATGCCCCACTCCAGCCTGCASTING‐SalI‐Flag‐R: ATCCAGAGGTTGATTGTCGACTCACTTGTCGTCATCGTCTTTGTAGTC AGAGAAATCCGTGCGGAGAGSTING‐C88A‐F: TGGAGGACTGTGCGGGCCGCCCTGGGCTGCCCCCTCCGSTING‐C88A‐R: CGGAGGGGGCAGCCCAGGGCGGCCCGCACAGTCCTCCASTING‐C91A‐F: TGCGGGCCTGCCTGGGCGCCCCCCTCCGCCGTGGGGCCCSTING‐C91A‐R: GGGCCCCACGGCGGAGGGGGGCGCCCAGGCAGGCCCGCASTING‐C88A/C91A‐F: TGGAGGACTGTGCGGGCCGCCCTGGGCGCCCCCCTCCGCCGTGGGGCCCSTING‐C88A/C91A‐R: GGGCCCCACGGCGGAGGGGGGCGCCCAGGGCGGCCCGCACAGTCCTCCASTING‐P141A‐F: CAAGGGCCTGGCCGCAGCTGAGATCTCTGCAGTGSTING‐P141A‐R: CACTGCAGAGATCTCAGCTGCGGCCAGGCCCTTGSTING‐Y199C‐F: AGTGAGCCAGCGGCTGTGTATTCTCCTCCCATTGGSTING‐Y199C‐R: CCAATGGGAGGAGAATACACAGCCGCTGGCTCACTSTING‐R238A‐F: GCTGGCATCAAGGATGCGGTTTACAGCAACAGCATCTATGAGCSTING‐R238A‐R: GCTCATAGATGCTGTTGCTGTAAACCGCATCCTTGATGCCAGCSTING‐S366A‐F: AGCCTGAGCTCCTCATCGCTGGAATGGAAAAGCCCCTSTING‐S366A‐R: AGGGGCTTTTCCATTCCAGCGATGAGGAGCTCAGGCTSTING‐42‐BamHI‐F: GCAT GGATCCATGCACACTCTCCGGTACCTGGTSTING‐82‐BamHI‐F: GCAT GGATCCATGTGGAGGACTGTGCGGGCCTGSTING‐111‐BamHI‐F: GCAT GGATCCATGAATGCGGTCGGCCCGCCCTTSTING‐110‐SalI‐Flag‐R: GCATGTCGACTCACTTGTCGTCATCGTCTTTGTAGTCTGGGAGGGAGTAGTAGAAATSTING‐1/150‐SalI‐Flag‐R: GCATGTCGACTCACTTGTCGTCATCGTCTTTGTAGTCTTTTTCACACACTGCAGAGASTING‐151‐BamHI‐F: GCATGGATCCATGGGGAATTTCAACGTGGCCCAVDAC2‐HA‐EcoRI‐F (for pCDNA3):GCATGAATTCATGTATCCATATGATGTTCCAGATTATGCTGCGACCCACGGACAGACTTGVDAC2‐HA‐XbaI‐F (for pLenti‐GFP hygro):GCATTCTAGAATGTATCCATATGATGTTCCAGATTATGCTGCGACCCACGGACAGACTTGVDAC2‐SalI‐R: GCAT GTCGAC TTAAGCCTCCAACTCCAGGGVDAC2‐C103A‐F: CAATTGAAGACCAGATTGCTCAAGGTTTGAAACTGAVDAC2‐C103A‐R: TCAGTTTCAAACCTTGAGCAATCTGGTCTTCAATTGVDAC2‐C210A‐F: CAATTTATCAGAAAGTTGCTGAAGATCTTGACACTTVDAC2‐C210A‐R: AAGTGTCAAGATCTTCAGCAACTTTCTGATAAATTGVDAC2‐C227A‐F: GGACATCAGGTACCAACGCCACTCGTTTTGGCATTGVDAC2‐C227A‐R: CAATGCCAAAACGAGTGGCGTTGGTACCTGATGTCCVDAC2‐E84Q‐F: GAGTATGGTCTGACTTTCACACAAAAGTGGAACACTGATAACACTCTGVDAC2‐E84Q‐R: CAGAGTGTTATCAGTGTTCCACTTTTGTGTGAAAGTCAGACCATACTCVDAC2‐Q101K‐F: CAGAAATCGCAATTGAAGACAAGATTTGTCAAGGTTTGAAACTGACVDAC2‐Q101K‐R: GTCAGTTTCAAACCTTGACAAATCTTGTCTTCAATTGCGATTTCTGVDAC2‐S128A‐F: GAAAAGTGGTAAAATCAAGTCTGCTTACAAGAGGGAGTGTATAAACCVDAC2‐S128A‐R: GGTTTATACACTCCCTCTTGTAAGCAGACTTGATTTTACCACTTTTCVDAC2‐L136F‐F: CAAGAGGGAGTGTATAAACTTTGGTTGTGATGTTGACTTTGATTTTGCVDAC2‐L136F‐R: GCAAAATCAAAGTCAACATCACAACCAAAGTTTATACACTCCCTCTTGATP2A2‐BglII‐HA‐F: GCATAGATCTATGTATCCATATGATGTTCCAGATTATGCTGAGAACGCGCACACCAAGACATP2A2‐Flag‐SalI‐R: GCATGTCGACTCACTTGTCGTCATCGTCTTTGTAGTCAGACCAGAACATATCGCTAAGRP75‐BamHI‐F: GCAT GGATCC ATGATAAGTGCCAGCCGAGCGRP75‐SalI‐R: GCAT GTCGAC TTACTGTTTTTCCTCCTTTTGZDHHC7‐BamHI‐F: GCATGGATCCATGCAGCCATCAGGACACAGZDHHC7‐SalI‐R: GCAT GTCGAC TCACACTGAGAACTCCGGGCZDHHC7‐sh3‐resist‐F: AGTGATTTTTCACCTCCCATTACAGTAATCCTGTTGATCTTZDHHC7‐sh3‐resist‐R: AAGATCAACAGGATTACTGTAATGGGAGGTGAAAAATCACTZDHHC7‐C160S‐F: GAAAATGGATCATCACTCCCCGTGGGTGAACAATTZDHHC7‐C160S‐R: AATTGTTCACCCACGGGGAGTGATGATCCATTTTCRT‐PCR primers are listed below:STING1‐F: CACTTGGATGCTTGCCCTCSTING1‐R: GCCACGTTGAAATTCCCTTTTTHMOX1‐F: AAGACTGCGTTCCTGCTCAACHMOX1‐R: AAAGCCCTACAGCAACTGTCGCYB5R3‐F: TCTACCTCTCGGCTCGAATTGCYB5R3‐R: CCTTGTCATCATCGCTGGAGATTXNIP‐qPCR‐F: ATATGGGTGTGTAGACTACTGGGTXNIP‐qPCR‐R: GACATCCACCAGATCCACTACTZDHHC3‐F: CCACTTCCGAAACATTGAGCGZDHHC3‐R: CCACAGCCGTCACGGATAAAZDHHC7‐F: CCCAAAGGAAACGCTACGAAAZDHHC7‐R: CGCGCTCGGGTTTAATACAGRNA18S‐F: TGCGGAAGGATCATTAACGGARNA18S‐R: AGTAGGAGAGGAGCGAGCGACCU6‐qPCR‐F: CTCGCTTCGGCAGCACAU6‐qPCR‐R: AACGCTTCACGAATTTGCGTPrimers used in generating plasmids used in this study are listed below:STING, cGAS, ZDHHC3, and ZDHHC7 were purchased from Sigma. Their target sequence is listed below:shScr: CCGGAACAGTCGCGTTTGCGACTGGCTCGAGCCAGTCGCAAACGCGACTGTTTTTTTTGshSTING‐1: CCGGCCAACATTCGCTTCCTGGATACTCGAGTATCCAGGAAGCGAATGTTGGTTTTTTGshSTING‐2: CCGGGCAGAGCTATTTCCTTCCACACTCGAGTGTGGAAGGAAATAGCTCTGCTTTTTTGshSTING‐3: CCGGGTCCAGGACTTGACATCTTAACTCGAGTTAAGATGTCAAGTCCTGGACTTTTTTGshcGAS‐1: CCGGCGTGAAGATTTCTGCACCTAACTCGAGTTAGGTGCAGAAATCTTCACGTTTTTTGshcGAS‐2: CCGGATCTATTCTCTAGCAACTTAACTCGAGTTAAGTTGCTAGAGAATAGATTTTTTGshZDHHC3‐10: CCGGGTATAGCATCATCAACGGAATCTCGAGATTCCGTTGATGATGCTATACTTTTTTGshZDHHC3‐18: CCGGGCTTTGAAGAAGATTGGACAACTCGAGTTGTCCAATCTTCTTCAAAGCTTTTTTGshZDHHC3‐79: CCGGCCAGAAGTACTTCGTCCTGTTCTCGAGAACAGGACGAAGTACTTCTGGTTTTTTGshZDHHC7‐3: CCGGGATAACTGTAATCCTGTTGATCTCGAGATCAACAGGATTACAGTTATCTTTTTTGshZDHHC7‐34: CCGGACTGCCCGTGGGTGAACAATTCTCGAGAATTGTTCACCCACGGGCAGTTTTTTTGshRNA plasmids to deplete endogenous shMCU: CCGGGCAAGGAGTTTCTTTCTCTTTCTCGAGAAAGAGAAAGAAACTCCTTGCTTTTTGα: CCGGCCAGTTATGATTGTGAAGTTACTCGAGTAACTTCACAATCATAACTGGTTTTTTGshHIF‐1α: CCGGGGAGACGGAGGTGTTCTATTTCAAGAGAATAGAACACCTCCGTCTCCTTTTTTGshHIF‐2shGRP75‐51:CCGGGCACATTGTGAAGGAGTTCAACTCGAGTTGAACTCCTTCACAATGTGCTTTTTTGshGRP75‐52:Other shRNA plasmids were constructed by inserting synthesized shRNAs into pLKO‐puro or pLKO‐hydro vectors. Their target sequence is listed below:shVDAC2: CCGGAAGGATGATCTCAACAAGAGCCTCGAGGCTCTTGTTGAGATCATCCTTTTTTTTGTet‐inducible shRNA plasmids were constructed by inserting synthesized shRNAs into Tet‐pLKO‐neo vector. Target sequence of STING was the same as that of shSTING‐2 and ‐3. Target sequence of shVDAC2 is listed below:sgSTING‐1B: GCTGGGACTGCTGTTAAACGsgSTING‐6: CATTACAACAACCTGCTACGsgcGAS‐1: CACGTGCTCATAGTAGCTCCsgcGAS‐2: GGCCGCCCGTCCGCGCAACTsgIRF3‐1: CGCTCACTGCCCAGTATGTGsgIRF3‐4: GGCACCAACAGCCGCTTCAGsgMCU: GTGTTTTCTAGGTACACCAGsgRNA plasmids were constructed by inserting synthesized sgRNAs into lentiCRISPRv2‐puro vector. Their target sequence is listed below:STING‐S366A‐KI‐sgF: CACCGGCTTTTCCATTCCACTGATGSTING‐S366A‐KI‐sgR: AAACCATCAGTGGAATGGAAAAGCCSTING‐S366A‐KI‐ssODNAGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTATTAATCGCTGGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTTGAGACCCAGGGTCACCAGGCCAGAGCCTCCSTING‐S366A knockin experiment was performed using STING sgRNAs and ssoDNA as listed below:STING‐S366A‐KI‐PCR‐F: GAGTGGGAATGGGTAAGATCCTCSTING‐S366A‐KI‐PCR‐R: GACGCATCTTAAGATGTCAAGTCCKnockin clones were screened by PCR using primers listed below to search for clones loss of SacI but gain of AseI site after knockin. Equal amounts of whole cell lysates were resolved by SDS‐PAGE and immunoblotted with indicated antibodies. For immunoprecipitations analysis, unless specified, 1 mg lysates were incubated with the indicated antibody (1–2 µg) for 3–4 h at 4 °C followed by 1 h incubation with 10 µL Protein A/G XPure Agarose Resin . Or 1 mg lysates containing tagged molecules were incubated with agarose beads coupled antibodies for the specific tag for 3–4 h at 4 °C. For endogenous IPs, incubation of cell lysates with antibodies was extended to overnight. The recovered immuno‐complexes were washed five times with NETN buffer  before being resolved by SDS‐PAGE and immunoblotted with indicated antibodies. For VDAC2 oligomerization detection, cells were incubated with 250 µM ethylene glycol bis (succinimidyl succinate) , lyzed in Triton X‐100 buffer and nixed with 4 × lithium dodecyl sulfate sample buffer . Load protein without boiling in SDS‐free PAGE gel and immunoblotted with indicated antibodies.Cells were lysed in EBC buffer  or Triton X‐100 buffer  supplemented with protease inhibitor cocktail  and phosphatase inhibitor cocktail . The protein concentrations of whole cell lysates were measured by NanoDrop OneC using the Bio‐Rad protein assay reagent as described previously.71 Equ 1‐day post‐transfection, cells were selected with 1 µg ml−1 puromycin for 3 days. Surviving cells were counted and each single cell was seeded into 96‐well plates. Each single clone grown up in 96‐well plates was amplified and one copy was used for genomic DNA extraction, followed by PCR and AseI/SacI digestion to screen for potential knockin clones. SacI negative but AseI positive clones are selected and sequenced to verify the knockin at the DNA level.Parental A498 cells were split into 24‐well plates and transfected with sgRNA against endogenous STING together with STING‐S366A‐ssoDNA following protocols as described.60 1‐dm urea  to 100 µg µl−1 and subjected to FASP trypsin digestion protocol. Briefly, proteins were reduced using 50 mM DTT  for 15 min at 65 °C. Proteins were then transferred to a 30 000 MWCO spin filter , diluted with 200 µl of 8 m urea, and centrifuged at 10 000 × g for 30 min at RT. The proteins were washed twice using 200 µl of 8 m urea followed by 30 minutes of centrifugation at 10 000 × g between each wash. Proteins were alkylated using 100 µl of 15 mM 2‐chloroacetamide  prepared in 8 m urea for 20 min in the dark at RT. The spin filter was then centrifuged at 10 000 × g for 20 min at RT followed by two washes with 200 µl of 8 M urea with 20 min of centrifugation at 10000 × g in between each wash. Buffer exchange into 50 mM ammonium bicarbonate (ABC) pH 8.0 was performed with two washes of 200 µl of 50 mM ABC with centrifugation at 10 000 × g for 15 min at RT between each wash. 100 µl of 50 mM ABC was added to the spin filter along with 2.5 µg of trypsin (Promega V511C). Proteins were trypsinized overnight at 37°C for 18 h. Following trypsinization, peptides were recovered in a new receiver tube by centrifugation at 10 000 × g for 15 min. Peptides were eluted twice using 50 µl of 0.5% TFA in water at 10 000 × g for 10 min. Samples were then concentrated to 100 µl using a speedvac followed by C18 desalting . Samples were then concentrated using a speedvac and resolubilized in 100 µl of LC‐Optima MS‐grade water (Thermo). Ethyl acetate extraction followed by speedvac was performed to remove residual detergents. QFP assay  was performed for peptide quantification.A498 cells stably expressing either FLAG‐EV or FLAG‐STING were lysed in EBC lysis buffer FLAG immunoprecipitants were washed with NETN buffer. Proteins were diluted in 8  Data was searched using MaxQuant (version 1.6.6.7), and all statistical analyses were done in Perseus (version 1.6.3.4).Detailed liquid chromatography‐mass spectrometry/mass spectrometry methods and data filtering methods were described previously.76 DatRNA extraction was performed with an RNA miniprep super kit  and QIAshredder . The final elution step was done with 50 µL of RNAse‐free water. The relative enrichment of mRNA was quantified with the NanoDrop OneC (ThermoFisher Scientific). At least two biological replicates were performed for RNA extraction. Reverse transcription was performed with iScript cDNA synthesis kit . Quantitative real‐time PCR was performed with iTaq universal SYBR green supermix  using a QuantStudio 6 Flex Real‐Time PCR Systems (ThermoFisher Scientific). Each mRNA level was normalized to RNA18S or U6 snRNA. The comparative Ct method was used to calculate fold change in expression. Statistical significance was determined by one‐way ANOVA tests.2) for 3 h. After thorough mixing, absorbance at 450 nm was measured using the BioTek Cytation 5 Cell Imaging reader. Combination index (CI) of co‐treatment of 2‐BP and sorafenib was calculated by CompuSyn software .Indicated number of cells were seeded in each well of 96‐well plates for cell viability assay to monitor cell viability at indicated time periods using Cell Counting Kit 8  according to manufacturer's instruction. Briefly, at indicated time points post‐cell seeding, 10 µL CCK8 solution was added into each well and incubated in the culture incubator (37 °C with 5% CO2 for 7–15 days (as indicated in figure legends) until formation of visible colonies. Colonies were washed with 1x PBS, fixed with methanol for 30 min, and stained with 0.5% crystal violet for 30 min. Colonies were then washed with distilled water and air‐dried. Colony numbers were manually counted. Three independent experiments were performed to generate the error bars.Indicated cells were seeded into 6‐well or 24‐well plates (500 cells/well) and cultured in 37 °C incubator with 5% CO Briefly, the assays were preformed using 6‐well plates where the solid medium consists of two layers. The bottom layer contains 0.8% noble agar, and the top layer contains 0.4% agar suspended with 3 × 104 or indicated number of cells. 500 µL complete DMEM medium with 10% FBS was added every 4 days. About 4–6 weeks later the cells were stained with iodonitrotetrazolium chloride (1 mg mL−1) (Sigma I10406) overnight for colony visualization, imaging, and counting. Three independent experiments were performed to generate the error bar.The anchorage‐independent cell growth assays were performed as described previously.53 Bri Briefly, for mouse xenograft growth experiments, A498 cells were infected with viruses expressing shScr or shSTING2/3. Two days later, 1 × 106 A498 cells in PBS were injected into the flank of indicated female nude mice . Tumor size was measured every two days with a digital caliper, and the tumor volume was determined with the formula: L × W2 × 0.5, where L is the longest diameter and W is the shortest diameter. After 66 days, mice were sacrificed, and tumors were dissected and weighed. For inducible STING shRNA, 1 × 107 A498 cells in DMEM were injected into flank of male nude mice . When tumors became visible after 15 days, 2 mg ml−1 doxycycline was added to drinking water containing 2% sucrose. Water was changed every three days and protected from light. Mice were sacrificed 39 days after injection. For combination therapy, 1 × 107 A498 cells in DMEM were injected into flank of male nude mice . After 25 days, when tumors reached a volume of ≈150 mm3, mice were randomly divided into four groups. 2‐BP was dissolved in 10% DMSO+40% PEG300+5% Tween 80+ 45% saline. Sorafenib was dissolved in 3% DMSO+97% sulfobutylether‐β‐Cyclodextrin solution . Mice were daily treated with 40 mg kg−1 2‐BP given by intraperitoneal injection or 40 mg kg−1 sorafenib given by oral gavage or both drugs or their solvents. Tumor size was measured regularly, and the tumor volume was determined with the formula: L x W2 x 0.5, Mice were sacrificed after 13 days of treatment.All mouse work has been reviewed and approved by UNC Institutional Animal Care and Use Committee under IACUC#19‐031 which has been continued as #22‐056. Mouse xenograft assays were performed as described previously.71 Bri Reads overlapping blacklisted regions of the genome were then removed. Transcript abundance was then estimated using Salmon, and differentially expressed genes were detected using DESeq2 with the criteria of adjusted p‐values (adjP) <0.05. The ClusterProfiler R package (v3.14.3) was employed to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional pathway annotation. Enrichment analysis for GO terms and KEGG pathways utilize enrichGO and enrichKEGG functions and visualizes the result with bubble plots. RNA‐seq data are deposited to GEO under accession number GSE190816.Total RNA from triplicate samples of A498 cells infected with shScr or shSTING was extracted with RNeasy mini kit . Library preparation and sequencing were performed by GENEWIZ as paired‐end 150‐bp reads. Reads were then filtered for adaptor contamination using cutadapt and filtered such that at least 90% of bases of each read had a quality score > 30. Reads were aligned to the reference genome (hg19) using STAR version 2.5.2b retaining only primary alignments.77 Rea4, 1 µM Alexa 647‐azide (Life Technologies), and 100 mM ascorbic acid (fresh) for 30 min. at room temperature in the dark. Finally, cells were resuspended in 1% BSA‐PBS with 1 µg mL−1 DAPI (Life Technologies) and 100 µg mL−1 RNAse A (Sigma Aldrich) and incubated overnight at 4 °C in the dark. Data were collected on an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using FCS Express 7 software (De Novo Software).To measure cell cycle phase distribution, cells were treated with 10 µM EdU  for 30 min. before harvesting by trypsinization. Cells were washed with PBS and then fixed in 4% paraformaldehyde in PBS for 15 min. at room temperature. 1% BSA‐PBS was added, mixed, and cells were centrifuged. Fixed cells were permeabilized with 0.5% triton X‐100 in 1% BSA‐PBS at room temperature for 15 min. and centrifuged. Cells were then processed for EdU detection as follows: samples were incubated in PBS with 1 mM CuSOTo measure general ROS level, trypsinized cells were incubated with 10 µM CM‐H2DCFDA  in PBS at 37 °C for 30 min. To measure mitochondrial ROS level, trypsinized cells were incubated with 1 µM MitoSOX Red mitochondrial superoxide indicator  in PBS at 37 °C for 20 min. To measure mitochondrial calcium level, trypsinized (without EDTA) cells were incubated with 5 µM Rhod‐2, AM  in PBS solution containing 10% FBS and 10 mM glucose at 37 °C for 1 h. To measure mitochondrial membrane potential, cells were incubated with 2 µM JC‐1  in PBS at 37 °C for 15 min. Cells were washed with PBS twice and analyzed with BD FACScanto II or BD FACSfortessa (BD Biosciences) with the BD Diva software (BD Biosciences). A minimum of 10 000 events were acquired for each sample. Data were analyzed using Flowjo 7.6.1 (Tree Star).Cells plated onto glass coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.2% Triton X‐100 for 20 min at room temperature. Cells were incubated with blocking buffer  for 1 h, incubated with primary antibodies at 4 °C overnight, incubated with secondary antibodies and/or mitochondria dye MitoView Green  at room temperature for 1 h and mounted with ProLong Gold antifade reagent . Fluorescent signals were observed with an Olympus 1 × 51 inverted microscope at × 20 magnification, or with an Olympus FV1000 confocal microscope at 60× magnification.−1. Added 800 µl reagent C and invert tubes several times to mix. Centrifuged tubes at 700 × g for 10 min and collect supernatant as a mixture containing mitochondria, lysosome, and peroxisome. Centrifuged supernatant at 3000 × g for 15 min and this pellet contains isolated mitochondria. Centrifuged supernatant at 12 000 × g for 15 min and transferred new supernatant containing cytosolic fraction to a new tube. The pellet was a mixture of lysosome, peroxisome, and some mitochondria. Resuspended mitochondria pellet in 500 µl reagent C and centrifuged at 12 000 × g for 5 min to obtain pure mitochondrial fraction. Samples were kept at 4 °C throughout the procedure.Cytosolic and mitochondrial fractions of cells were isolated with Mitochondria isolation kit  for cultured cells according to manufacturer's instruction. Briefly, add 800 µl reagent A to cell pellets and incubate for 2 min. Added 10 µl reagent B and vortex 5 s min Image analysis was performed using Adobe Illustrator.Electron microscopic analysis of indicated A498 cells was performed as detailed below. Sample prep: indicated A498 cell culture pellets were embedded in 2% agarose, secondary fixation with osmium tetroxide, dehydration , followed by embedding in EPON. Sectioning: ≈80 nm thin sections on Cu grids, stained with UA and LC. Imaging: FEI Tecnai 12 at 120 kV, Gatan Rio16 CMOS camera. Cells were randomly selected for imaging. Images at 4400× magnification were used for counting total and abnormal mitochondria. All clearly identified mitochondria in the images were analyzed. Images at 26 000× magnification were randomly selected for mitochondria‐ER contact (MERC) length measurement. All clearly identified mitochondria membranes and associated ER membranes in the images were evaluated. Only MERC sites with a distance no more than 30 nm were regarded as MERC enabling calcium transfer from ER to mitochondria and their length was measured according to.80 Ima4 cells were seeded into XF24 cell culture microplate  before the assay. Added 1 mL of Seahorse XF24 calibrant solution (pH 7.4)  to each well of XF24 microplate and replaced green sensor cartridge on the top. Incubated entire cartridge in a non‐CO2 incubator at 37°C overnight. On the day following cell seeding, media was changed to phenol red‐free, Seahorse XF DMEM medium, pH 7.4  supplemented with 25 mM glucose and 1 mM sodium pyruvate for OCR or 2 mM glutamine and 1 mM sodium pyruvate for ECAR. Cells were equilibrated within 1 h in 37 °C non‐CO2 incubator. Loaded the sensor cartridge for the calibration process and replaced calibration plate with cell plate after that. The basal mitochondrial respiration or extracellular acidification rate was first measured by recording extracellular oxygen concentration. Then the OCR or ECAR trace was recorded in response to sequential addition of indicated compounds in the Seahorse XF Cell Mito Stress Test Kit  or Glycolysis Stress Test Kit . Concentration of compounds for OCR was: 1 µM of oligomycin A, 1 µM of FCCP, and 1 µM of rotenone/antimycin A. Concentration of compounds for ECAR was: 10 mM of glucose, 1 µM of oligomycin, and 50 mM of 2‐DG. 3 to 5 technical replicates were utilized per sample to calculate OCR or ECAR.The OCR and ECAR were measured by an XFe24 extracellular flux analyzer (Agilent Technologies), according to the manufacturer's instructions. Briefly, 5 × 10 Briefly, cells were lysed in lysis buffer (LB)  supplemented with protease inhibitor, PMSF and 50 mM N‐ethylmaleimide (NEM) . Equal amounts of lysates were incubated with the STING antibody overnight at 4 °C followed by 1 h incubation with 60 µL Protein A/G XPure Agarose Resin, or incubated with Flag agarose beads for 3–4 h at 4 °C. Beads were resuspended in LB+10 mM NEM and split into triplicates. 1/3 of beads were used as ‐HAM control and 2/3 were used as +HAM treatment. Beads were resuspended with LB (pH 7.2)+0.1% SDS quickly, washed with LB (pH = 7.2) three times, resuspended in LB (pH 7.2) with or without 1 m hydroxylamine (HAM)  and incubated at room temperature for 1 h. Then, beads were washed with LB (pH 6.2) once and resuspended in LB (pH = 6.2)+2 µM Biotin‐BMCC  at 4 °C for 1 h. Beads were then washed with LB (pH = 6.2) once and LB (pH 7.5) for three times. Beads were boiled and resolved by SDS‐PAGE and immunoblotted with streptavidin‐HRP antibody to detect biotin‐labeled palmitoylated STING.Protein palmitoylation detection was performed by immunoprecipitation and acyl‐biotin exchange as described previously.81 Brip ≤ 0.05 was considered statistically significant. The results were shown as means ± SD from at least two or three independent experiments as indicated in figure legends. Differences between control and experimental conditions were evaluated by One‐way ANOVA.Statistical analyses were performed using the Graphpad Prism 8 Software. The authors declare no conflict of interest.Conceptualization: ZZ, PL; Methodology: ZZ, JGC, BM, XT, HY, GD, PL; Investigation: ZZ, HD, XZ, EWC, JZ, LM, YW, XT, TS, JS; Visualization: ZZ, YW, JZ; Funding acquisition: JGC, DH, GD, PL; Project administration: JLS, EH; Supervision: JGC, BM, GD, PL; Writing – original draft: ZZ, PL; Writing – review & editing: HY, JGC, BM, GD.Supporting InformationClick here for additional data file.


The crystal structure of the title salt is held by N—H⋯Br charge-assisted hydrogen bonds and dipole–dipole interactions. In comparison with reference structures, elongation of C—S bonds is observed. 12H13N2S+·Br−, which crystallizes in the monoclinic P21/c centrosymmetric space group. The asymmetric unit contains one 2-[methyl]isothiouronium cation and one bromide anion. The methylene carbon lies in plane of the naphthalene core. In comparison with reference structures, elongation of C—S bonds as well as tilting of the isothiouronium group is observed. Given the ionic nature of the compound, the structure is held by charge-assisted N—H⋯Br hydrogen bonds, with a noteworthy contribution of dipole–dipole interactions, which form bilayers in the structure. The bilayers are held by the weak London forces.Herein we report the crystal structure of 2-[methyl]isothiouronium bromide, C Since the relevant torsion angle of the title compound is −68.1 (3)°, only the non-linear 2-benzylisothiouronia, will be used as a reference group [CCDC codes EBIFOK methyl]isothiouronia structures in the the CSD; therefore, we decided to compare the title compound with 2-benzylisothiouronia structures. The published structures of 2-benzylisothiouronia can be divided according to the CThe C11—S1 bond is almost perpendicular to the naphthalene core, with C1—C2—C11—S1 and C3—C2—C11—S1 torsion angle values of 91.8 (3) and −88.0 (3)°, respectively. Among the reference group, such a conformation is unusual, with average values being 61.45 and 120.53°. The isothiouronium group is significantly tilted, with C11—S1—C12—N1 and C11—S1—C12—N2 torsion angles of −68.5 (3) and 110.7 (3)°, respectively. Such a tilt is not observed among the reference group, where the average values are 20.46 and 160.94°. The tilting of the group can be explained by the steric demands of the 2-naphthylmethyl unit on the packing.n1⋯Br1⋯H2n1iii—N1iii, and N2—H1n2⋯Br1ii⋯H2n2ii—N2ii classified as n1⋯Br1⋯H1n2iv—N2iv—C12iv—N1iv, N1—H2n1⋯Br1i⋯H1n2v—N2v—C12v—N1v, and N1—H2n1⋯Br1i⋯H2n2i—N2i—C12i—N1i classified as x, y − 1, z; (ii) x, −y + z − x, y + 1, z; (iv) x, −y + z + x, −y + z + vi isothiouronium bromide.The 2-[methyl]isothiouronium bromide (20 mg) was dissolved in 10 ml of methanol and left to slowly evaporate at room temperature. After 5 d, colorless platelets were collected.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620015114/sj4217sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314620015114/sj4217Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620015114/sj4217Isup3.molSupporting information file. DOI: 2044275CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The coordination sphere around each Cd2+ ion is completed by three N atoms of a tridentate 8-[2-(dimethylamino)ethylamino]quinoline ligand and another chloride ion to form a distorted fac-CdN3Cl3 octahedron. The ethanol molecule is both an acceptor of an N—H⋯O and a donor of an O—H⋯Cl hydrogen bonds to its adjacent complex unit. In the crystal, weak aromatic π–π stacking is observed.The title solvated bimetallic complex, [Cd Similar perpendicular arrangement of the pendant Cl− ions is observed in the Cl–(Cd2Cl2)–Cl fragments of other complexes with different ligands ° (N3—Cd1—N2) to 105.73 (3)° (Cl1—Cd1–Cl2) for one Cd2+ ion and 71.04 (9) ° (N6—Cd2—N5) to 102.09 (7)° (N5—Cd2—Cl2) for the other. The corresponding angles for the hydrate structure are in the range 69.48 (5) to 101.08 (4)°. The N—C—C—N torsion angles in the ethane diamine are almost the same for both independent ligands [N1—C3—C4—N2 = 63.0 (4)° and N4—C16—C17—N5 = 63.3 (5)°] in I.Both CdR(6)22 loop. In the extended structure, the quinoline ring systems of neighbouring complex units are involved in weak aromatic π–π stacking interactions. The groups involved are related by inversion symmetry with a c(i)⋯c(i)′ separation of 3.93 (1) Å [c(i) = the midpoint of the C9—C10 bond of the C5–C13/N3 ring system]. If a second longer inversion-related contact c(ii)⋯c(ii)′ of 4.56 (1) Å [c(ii) = midpoint of the C22—C23 bond of the C18–C26/N6 ring system] is considered to be a significant interaction, infinite chains running parallel to [101] result ethylamino]quinoline ligand and cadmium dichloride were mixed in dry ethanol solvent at room temperature under a positive nitrogen pressure and the mixture was stirred at room temperature for several hours. The solution was then warmed to dissolve the material and the product was recrystallized on cooling to produce colourless crystals of Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621001504/hb4375sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621001504/hb4375Isup2.hklStructure factors: contains datablock(s) I. DOI: 2062006CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure of a resveratrol–oxime ester is reported. 22H25NO6, the C=C double bond linking the benzene rings adopts an E configuration and the dihedral angle between the rings is 47.1 (2)°. The oxime unit contains a C=N double bond, which also has an E configuration. In the crystal, pairs of C—H⋯N hydrogen bonds generate inversion dimers and weak C—H⋯O interactions link the dimers into chains propagating along the b-axis direction.In the title compound, C The dihedral angle formed by benzene rings is 47.1 (2)°. The C17=N1 imine double bond in the oxime unit also adopts an E configuration, which is defined by a torsion angle of 178.3 (1)° for C4—C17—N1—O3. There are three methoxy groups attached to carbon atoms C1, C5 and C13 in the benzene rings: those at the meta positions  are essentially co-planar with their attached benzene rings [C6—C1—O1—C7 = −0.2 (2)° and C6—C5—O6—C22 = 3.9 (2)°] whereas the methoxy group at the para position (C13) is slightly twisted from the corresponding ring plane [C12—C13—O2—C16 = 8.9 (2)°]. In the crystal, pairs of C22—H22⋯N1 hydrogen bonds generate inversion dimers benzaldehyde  I. DOI: 10.1107/S2414314621009500/hb4391Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621009500/hb4391Isup3.cmlSupporting information file. DOI: 2109339CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Using the equation-of-motion coupled-cluster singles and doubles method and Fermi’s golden rule, we quantitatively reproduced all rate constants relevant to the emission mechanism; prompt and delayed fluorescence, internal conversion (IC), intersystem crossing, and reverse intersystem crossing (RISC). In addition, the photoluminescence quantum yield and its prompt and delayed contributions were quantified by calculating the population kinetics of excited states and the transient photoluminescence decay curve. The calculations also revealed that TADF occurred via a stepwise process of 1) thermally activated IC from the electronically excited lowest triplet state T1 to the second-lowest triplet state T2, 2) RISC from T2 to the lowest excited singlet state S1, and 3) fluorescence from S1.Molecules that exhibit multiple resonance (MR) type thermally activated delayed fluorescence (TADF) are highly efficient electroluminescent materials with narrow emission spectra. Despite their importance in various applications, the emission mechanism is still controversial. Here, a comprehensive understanding of the mechanism for a representative MR-TADF molecule  are highly efficient electroluminescent materials, but their emission mechanism is still not fully understood. Here, the emission mechanism of a representative MR-TADF molecule is studied through quantum chemistry calculations, with two consecutive decay processes unveiled. An important factor for TADF efficiency is the energy difference between the lowest triplet state (T1) and the lowest excited singlet state (S1), ∆E(T1 → S1); a small ∆E(T1 → S1) (<200 meV) is required to induce T1 → S1 reverse intersystem crossing (RISC). Another important factor is a large transition-dipole moment between S1 and ground-state S0 to accelerate the rate of S1 → S0 fluorescence. Experimentally, efficient RISC and high photoluminescence quantum yields (PLQYs) have been simultaneously realized by combining suitable donor and acceptor units that control the spatial overlap between the highest-occupied molecular orbitals (HOMOs) and the lowest-unoccupied molecular orbitals (LUMOs)9. It is important to note that the fluorescence spectra were broadened because of the charge-transfer character of S1, which was a significant drawback for OLED applications in displays.Since highly efficient thermally activated delayed fluorescence (TADF) was used in organic light-emitting diodes (OLEDs)10. Using a triphenylboron core possessing two nitrogen atoms . However, ∆E(T1 → S1) and ∆E(T1 → T2) were large, 0.59 eV and 0.61 eV, respectively, which could not explain efficient RISC from T1 to S1. Gao et al. examined the density-functional dependence of ∆E(T1 → S1) within the framework of TD-DFT41. The MPWK1CIS functional reproduced the experimental ∆E(T1 → S1)41, as reviewed by Suresh et al.21. However, the RISC rate constant (kRISC) was not calculated and the TADF mechanism was unclear. Also, they considered TADF in terms of direct (one-step) T1 → S1 RISC, which differed from the work of Northey et al40. Pershin et al. reported that TD-DFT methods overestimated ∆E(T1 → S1) for MR molecules, and that the spin-component-scaling second-order approximate coupled-cluster (SCS-CC2) method outperformed TD-DFT for predicting ∆E(T1 → S1)43. The partial inclusion of double excitations within the SCS-CC2 method was responsible for the improved accuracy in predicting ∆E(T1 → S1). The SCS-CC2 calculation of DABNA-1-based molecules revealed the relationship between its molecular structure and electronic properties, ∆E(T1 → S1), and the fluorescence rate constant. However, the TADF mechanism was still unclear because the kRISC was not calculated. Lin et al. calculated rate constants for fluorescence (kF), ISC (kISC), kRISC, and internal conversion (IC) (kIC) for DABNA-144. However, the calculated kRISC was 6.7 × 102 s−1, which was much less than the experimental value of 1.0 × 104 s−110. The calculated kISC of 1.4 × 104 s−1 was two orders of magnitude less than the experimental value of 4.5 × 106 s−110. In addition, the calculated nonradiative decay, kIC(S1 → S0), was greater than the kF(S1 → S0), suggesting that the PLQY of DABNA-1 was less than 50%, in contrast with the 88% experimental value10. All these previous studies44 only partially explained the photophysical properties of MR-TADF mainly because crucial RISC was not elucidated.Several theoretical studies have attempted to reveal the TADF mechanism in MR molecules. Northey et al. investigated an intersystem-crossing (ISC) mechanism in DABNA-1 using quantum dynamics and time-dependent density-functional theory (TD-DFT) at the PBE0/6-31 G(d) levelE(T1 → S1) (and spin–orbit coupling (SOC) in some cases) and oscillator strength have been considered to understand the TADF mechanism and to design TADF molecules. However, these are not sufficient for the above aim; quantifications of all types of rate constants and those of energy levels, including higher-lying states relevant to the emission process, are required for the complete understanding of the emission mechanism. Recently, we reported rate-constant predictions enabled by a proposed cost-effective calculation method based on Fermi’s golden rule45. Using the method, we calculated kIC, kISC, kRISC, kF, and the rate constant for phosphorescence (kPhos), of benzophenone as an example. The calculated rate constants agreed well with experimental values. Here, we applied the same method to DABNA-1 and determined all relevant rate constants. We also determined the lifetime of the delayed component in transient PL (trPL), the rate constant of TADF (kTADF), and the PLQY, by calculating the population kinetics of the excited states and the trPL decay curve. The calculations also indicated that, after photoexcitation, T1 was first generated via S1 → T2 ISC and T2 → T1 IC. Then, triplet-to-singlet conversion occurred via thermally activated T1 → T2 IC and T2 → S1 RISC.Here, we report a comprehensive understanding of the TADF mechanism using the representative MR molecule, DABNA-1. So far, ∆0, S1, T1, and T2 geometries of DABNA-1 were obtained at the TPSSh/6-31+G(d) level. Then, excited states were computed using the equation-of-motion coupled-cluster singles and doubles (EOM-CCSD) method (see “Methods” section). At the TD-TPSSh level, S1 and T1 local-energy minima were located at the S1- and T1-optimized geometries, respectively. However, as shown in Supplementary Table 1 and T1 energy levels calculated at the EOM-CCSD level were both located at the T1 geometry . In addition, the S1 and T1 structures were nearly the same, including the dihedral angles . Because S2 states were 0.89 eV higher in energy than the S1 states, their contributions were neglected  and that from T2 to T3 was smaller than that from T2 to T1  was only 0.06 eV larger than the experimentally obtained 0.2 eV. Thus, EOM-CCSD significantly improved the overestimation of ∆E(T1 → S1), relative to those by previously reported TD-DFT methods44.S1. Figure kIC, kISC, kRISC, kF, and kPhos. The raw rate constants in Table kIC, kISC, kRISC, kF, and kPhos are given in Supplementary Equations We examined triplet formation from SkF(S1 → S0) (1.4 × 108 s−1) and kIC(S1 → S0) (1.2 × 107 s−1) agreed quantitatively with experimental results  determined by Hatakeyama et al.10 from PL decay curves of a 1-wt% DABNA-1: 9,9′-biphenyl-3,3′-diylbis-9H-carbazole film at 300 K  agreed with kISC(S1 → T2) (5.4 × 106 s−1), rather than kISC(S1 → T1) (1.7 × 105 s−1), suggesting that the experimental kISC should be assigned to kISC(S1 → T2). The kISC(S1 → T2) was ten times greater than kISC(S1 → T1), because of the larger S1–T2 SOC (1.52 cm−1) relative to that of S1–T1 (0.06 cm−1). The large S1 → T2 SOC enhanced S1 → T2 ISC, despite the uphill transition from S1 to T2 . The small S1–T1 SOC resulted from very similar S1 and T1 orbital configurations (HOMO → LUMO transition). These results suggested that the ISC (S1 → T1 conversion) of DABNA-1 occurred via the stepwise S1 → T2 → T1 process, rather than by the one-step S1 → T1 ISC, irrespective of the geometry relaxation  is sufficiently small46. Because T2 → T1 IC was much faster than S1 → T2 ISC, the latter was the rate-determining step of the S1 → T2 → T1 process  was approximately equal to kISC (S1 → T2) = 5.4 × 106 s−1  (2.5 s−1) was considerably less than the experimental value (1 × 104 s−1)10, indicating that T1 → S1 conversion did not occur in one-step but via a more effective process. Because the S2 and T3 energy levels, as well as higher singlet and triplet states, could not contribute to the RISC from T1, as discussed above, and T2 was close to S1 , we calculated the trPL decay curve  were then obtained from the trPL decay curve  were calculated fromISC) was calculated fromFor RISC from TT1 → S1) .5 s−1 waT1 → S1) .5 s−1 wakTADF was determined from10Then, k(T1 → T2 → S1) was calculated fromFinally, P, ΦTADF, ΦISC, τTADF, kTADF, k(T1 → T2 → S1), and k(S1 → T2 → T1) values. The Φ, ΦP, ΦTADF, and ΦISC values were quantitatively consistent with the experimental values because of the quantitative predictions for kF(S1 → S0), kIC(S1 → S0), kISC(S1 → T2), and kISC(S1 → T1). The calculated conversion rate constant k(S1 → T2 → T1) was consistent with the experimental 1 → T2 → T1 ISC rather than direct S1 → T1 ISC. All the calculated single-step rate constants and k(S1 → T2 → T1) agreed well with the experimental values. However, the agreements of the calculated kTADF and k(T1 → T2 → S1)  and the experiments  were not as good as we expected, compared with the case of k(S1 → T2 → T1). Considering that they included the uphill-energy T1 → T2 IC, there was a possibility that the overestimation of ∆E(T1 → T2) leads to an underestimation of kIC(T1 → T2), kTADF, and k(T1 → T2 → S1).Table E(T1 → T2) on kIC(T1 → T2), kTADF, and k(T1 → T2 → S1), we recalculated these rate constants using a corrected ∆E(T1 → T2). The rate-determining step for the T2-mediated RISC was T1 → T2 IC; hence, ∆E(T1 → T2) could be viewed as the activation energy for DABNA-1 TADF and RISC. As discussed above, the energy difference between the calculated and experimental T1 → S1 was 60 meV. Thus, 60 meV was subtracted from the EOM-CCSD-calculated ∆E(T1 → T2) value of 252 meV. The energy levels and rate constants calculated with the corrected ∆E(T1 → T2) are also listed in Table k(S1 → T2 → T1) was unchanged, suggesting that the T1 energy correction had little effect on the ISC. In contrast, kIC(T1 → T2) increased from 1.1 × 107 s−1 to 3.3 × 108 s−1. As a result, kTADF and k(T1 → T2 → S1) increased to 1.8 × 104 s−1 and 1.9 × 104 s−1, respectively, which were in much better agreements with the experiments. These results suggested that the ∆E(T1 → T2) overestimation was responsible for the underestimation of kTADF and k(T1 → T2 → S1). Thus, kTADF and k(T1 → T2 → S1) depended largely on kIC(T1 → T2), which was the sum of IC rate constants for individual molecular vibrations, kαIC,(T1 → T2), where α denoted the αth vibrational mode (Supplementary Equations −1) had the largest kαIC,(T1 → T2) value  method, whereas those of S1, T1, and T2 were performed using the TD-TPSSh/6-31+G(d) method with spin multiplicity = 1 -PCM model reproduced experimental DABNA-1 emission and absorption wavelengths in CH2Cl241. The geometry optimization and frequency analysis were performed with the Gaussian 16 program package47. For MR molecules, EOM-CCSD method and algebraic diagrammatic construction of the second order, have been shown to be suitable for calculating singlet–triplet energy differences50. Here, excited-state calculations were performed using the EOM-CCSD/6-31 G method implemented in the Q-Chem program package51. Excitation energies, SOCs, vibronic couplings, transition-dipole moments, and permanent dipole moments were calculated using EOM-CCSD wave functions. Examples of Q-Chem input files for running SOC calculations are shown in the Supplementary Methods.Geometry optimization and frequency analysis of SkIC is described elsewhere52 and reviewed in Supplementary Equation α and each α (Supplementary Equation S5). This protocol was also used for calculating the IC rate constants for Mathematical formulation of a method of calculating vibronic couplings and Supplementary InformationPeer Review File


To the Editor:TP53 encodes the tumour suppressor protein p53, a master regulator of genomic integrity and cell survival, and is the most frequently mutated gene.3‐p53m‐RA competitively abolished p53‐R175H pulldown from cell lysates with N3‐p53m‐RA‐biotin. By contrast, there was almost no pulldown of wild‐type p53 (p53‐WT)  Figure . Structu) Figure , but the) Figure . Moreove) Figure . TherefoA Figure .3‐p53m‐RA, indicating successful conjugation  for H1299‐p53‐R175H and SKBR3 cells were 1.06 and 1.28 µM, respectively  demonstrated a slight difference in dp53m‐RA migration relative to Nn Figure . Simulatn Figure . Indeed,n Figure , suggestn Figure . Time‐con Figure . To subsn Figure . The haly Figure . When thy Figure . The expy Figure . Furthery Figure . Moreovey Figure , and dp5y Figure . These fWe next examined the biological consequences of dp53m‐RA‐mediated p53‐R175H degradation in cancer cells. dp53m‐RA treatment dramatically inhibited the proliferation of p53‐R175H‐expressing H1299 cells and SKBR3 cells. By contrast, dp53m‐RA treatment did not affect the proliferation of p53‐WT‐expressing H1299 cells and A549 cells Figure . dp53m‐RTP53 mutation with dominant‐negative and oncogenic gain‐of‐function activities, as a binder in PROTAC design, we report the first selective mutant p53 degrader, dp53m‐RA. dp53m‐RA degraded p53‐R175H but not wild‐type p53 or other p53 mutants in a ubiquitin‐proteasome‐dependent manner. Importantly, dp53m‐RA inhibited the proliferation and migration of cancer cells specifically harbouring the p53‐R175H mutation (Figure In summary, by harnessing an RNA aptamer specifically targeting p53‐R175H, the most common n Figure , suggestThe authors declare they have no conflicts of interest.Supporting InformationClick here for additional data file.


E. The terminal benzene ring is approximately perpendicular to the central 1,4-diazabutadiene mean plane, forming a dihedral angle of 81.2 (3)°.The title molecule is disposed about a centre of inversion and the conformation about the imine bond is  52H40N2, is disposed about a centre of inversion and the conformation about the imine bond [1.268 (3) Å] is E. The terminal benzene ring is approximately perpendicular to the central 1,4-diazabutadiene mean plane, forming a dihedral angle of 81.2 (3)°. Weak C—H⋯π and π–π [inter-centroid distance = 4.021 (5) Å] interactions help to consolidate the packing.The title compound, C Å] has an anti-disposition and the imine bonds [1.268 (3) Å] are E-configured. The dihedral angle between the pendent benzene ring and the 1,4-diazabutadiene least-squares plane is 81.2 (3)°, consistent with an almost perpendicular relationship. In the crystal, C—H⋯π, Table 1x, 1 − y, 1 − z.In this study, we designed and synthesized the title compound Fig. 1 as a pot2O led to the corresponding triphenyl-substituted aniline  I, New_Global_Publ_Block. DOI: 10.1107/S2414314620005313/tk4062sup3.txtL1-cif. DOI: 1949863CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The main intermolecular interactions in the two title salts are O⋯H/H⋯O contacts, as revealed by Hirshfeld surface analyses. 5H5N4O+·C7H5O6S−·2H2O, (I), and 6-oxo-1,9-dihydropurin-7-ium perchlorate monohydrate, C5H5N4O+·ClO4−·H2O, (II), have been synthesized and characterized using single-crystal X-ray diffraction and Hirshfeld analysis. In both salts, the hypoxanthine molecule is protonated at the N7 position of the purine ring. In salt (I), the cation and anion are connected through N—H⋯O interactions. The protonated hypoxanthine cations of salt (I) form base pairs with another symmetry-related hypoxanthine cation through N—H⋯O hydrogen bonds with an R22(8) ring motif, while in salt (II), the hypoxanthine cations are paired through a water molecule via N—H⋯O and O—H⋯N hydrogen bonds with an R33(11) ring motif. The packings within the crystal structures are stabilized by π–π stacking interactions in salt (I) and C—O⋯π interactions in salt (II). The combination of several interactions leads to the formation of supramolecular sheets extending parallel to (010) in salts (I) and (II). Hirshfeld surface analysis and fingerprint plots reveal that O⋯H/H⋯O contacts play the major role in the crystal packing of each of the salts, with a 54.1% contribution in salt (I) and 62.3% in salt (II).Two salts of 1,9-dihydropurin-6-one (hypoxanthine), namely, 6-oxo-1,9-dihydropurin-7-ium 5-sulfosalicylate dihydrate, C When hypoxanthine interacts with strong acids, it becomes protonated at position N7 or N9. A limited number of hypoxanthine salts like hypoxanthine nitrate , a potential oxygen-free radical generator, is a strong agent against cancer cells (Susithra I), and hypoxanthinium perchlorate monohydrate, (II), salts and the noncovalent interactions that govern their crystal packings.The current article reports the crystal structures of hypoxanthinium 5-sulfosalicylate dihydrate, (2.I) crystallizes with two hypoxanthinium cations (A+ and B+), two 5-sufosalicylate anions  and four solvent water molecules  in the asymmetric unit, as shown in Fig. 1I), the B cation is equally disordered over two sets of sites for atoms C5B/C5C, C6B/C6C and O6B/O6C. Atoms H1B/H1C and H7B/H7C attached to N1B and N7B, respectively, are also disordered. The solvent water molecule O3W is also disordered over two positions. Atoms N7A and N7B are protonated, which is confirmed by widening of the C5A—N7A—C8A angle to 107.1 (4)° compared to the value of 103.8° in the two polymorphic forms of the neutral HX molecule ° and N3B—C4B—C5C—N7B = −178.3 (6)° are similar to those of the two forms of the neutral HX molecule ° and O7B—C9B—C10B—C11B = 175.9 (4)°], a situation that is likewise observed for previously reported crystal structures involving 5SCA− anions.Salt (II) crystallizes with one hypoxanthinium cation, one perchlorate anion (PCA−) and one solvent water molecule in the asymmetric unit. The molecular structure of salt (II) is shown in Fig. 2I). The PCA− anion has the characteristic tetrahedral shape, with Cl—O bond lengths between 1.4116 (15) and 1.4421 (15) Å, and O—Cl—O angles between 108.29 (9) and 111.24 (12)°.Salt (3.I), (010) sheets of cations and sheets of anions are stacked alternately along [010]. The crystal packing is governed by N—H⋯O, O—H⋯N and C—H⋯O hydrogen bonds  self assemble into sheets by interaction of symmetry-related counterparts through O7A—H7D⋯O10A and O7B—H7E⋯O10B, respectively  via N1B—H1C⋯O11A and C2B—H2B⋯O11A with an W—H1WA⋯O10B, O1W—H1WB⋯O12B, O2W—H2WA⋯N3A and O2W—H2WB⋯O12A  is consolidated by π–π interactions between the phenyl rings of the two 5SCA anions (C10A–C15A and C10B–C15B), and the imidazole ring (C4A–N9A) and the pyrimidine ring (N1A–C6A) of cation A+, with centroid-to-centroid distances of 3.547 (3), 3.562 (3), 3.554 (3) and 3.533 (3) Å, and slippages of 0.815, 1.300, 1.182 and 1.105 Å , (010) sheets of cations and sheets of anions are stacked alternately along [010]. The crystal packing of salt (II) is dominated by N—H⋯O and O—H⋯O hydrogen bonds, and to a minor extent by C—H⋯O hydrogen bonds  is shown in Fig. 8In the crystal structure of salt (s Table 2. The pro4.I) and (II) were performed using CrystalExplorer17  and 10(b) for (I) and (II), respectively. Corresponding two-dimensional fingerprint plots  and (II) are shown in Figs. 11I), 62.3% (II); N⋯H/H⋯N 3.1% (I), 6.8% (II); C⋯H/H⋯C 5.9% (I), 5.4% (II); H⋯H/H⋯H 16.0% (I), 5.3% (II); C⋯C/C⋯C 0.9% (I), 0.1% (II).Hirshfeld surface (HS) analyses of salts  and (II) with the related salt forms of guanine, xanthinium and hypoxanthine reveal that, in most of the crystal structures containing purine derivatives, the purine forms base pairs through pairs of N—H⋯O or N—H⋯N hydrogen bonds with an et al., 2014I) and (II) and related systems compiled in Table 3A comparison of salts (6.I) was synthesized by mixing an equimolar ratio of hypoxanthine (0.0340 g) and 5-sulfosalicylic acid (0.0545 g) in hot water. The solution was heated to 333 K for 1 h and then allowed to cool slowly to room temperature. Colourless needle-shaped crystals were harvested from the mother liquid after one week.Salt (II) was synthesized by mixing an equimolar ratio of hypoxanthine (0.0340 g) and iron perchlorate monohydrate (0.0681 g) in hot water. The solution was heated to 333 K with constant stirring for 1 h and then allowed to cool slowly to room temperature. Colourless plate-like crystals were harvested from the mother liquid after one week.Salt (7.I) and (II) are summarized in Table 4I), carbon (C5 and C6) and oxygen (O6) atoms of cation B are equally disordered over two sets of sites, with a refined occupancy ratio of 0.503 (18):0.497 (18). The solvent water molecule O3W is disordered over two positions, with a refined site-occupancy ratio of 0.58 (6):0.42 (6). The H atoms of water molecules O1W and O2W were located from a difference Fourier map, and the O—H distance restrained to 0.82 Å. Attempts to localize the H atoms of O3W and O4W in (I) from difference Fourier maps failed as there were no relevant electron densities close to these atoms. Hence, these H atoms are not part of the model but are included in the formula. All C- and N-bound H atoms in (I) were placed in idealized positions and refined freely using a riding model, with C—H = 0.95 Å and N—H = 0.86 Å, and with Uiso(H) = 1.2Ueq. In salt (II), the N-bound H atoms were located in a difference Fourier map and refined freely. The H atoms of the water molecule were likewise located from a difference Fourier map. The geometry of the water molecule was restrained using DFIX commands with an O—H distance of 0.85 Å and an H⋯H distance of 1.36 Å. All C-bound H atoms were treated as for salt (I).Crystal data, data collection and structure refinement details of salts  I, II, global. DOI: 10.1107/S2056989022004753/wm5640Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022004753/wm5640IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2170315, 2170314CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The 1,3-oxazolidine ring adopts an envelope conformation with the N atom in an endo position. The mean plane of the oxazolidine ring makes dihedral angles of 77.74 (10) and 45.50 (11)°, respectively, with the 4-bromophenol and 1,3,5-trimethylbenzene rings. In the crystal, adjacent molecules are connected via C—H⋯O hydrogen bonds and C—H⋯π interactions into layers parallel to the (200) plane. The packing is strengthened by van der Waals interactions between parallel molecular layers. A Hirshfeld surface analysis shows that H⋯H (58.2%), C⋯H/H⋯C (18.9%), and Br⋯H/H⋯Br (11.5%) interactions are the most abundant in the crystal packing.The title compound, C The mean plane of th oxazolidine ring makes dihedral angles of 77.74 (10) and 45.50 (11)°, respectively, with the 4-bromophenol (C6–C11) and the 1,3,5-trimethylbenzene (C14–C19) rings. The molecular conformation is stabilized by intramolecular O11—H11⋯N3 and C20—H20C⋯O1 hydrogen bonds  plane , C⋯H/H⋯C (18.9%), and Br⋯H/H⋯Br (11.5%) contacts are shown in Fig. 5b–d, while numerical details of the different contacts are given in Table 2A Hirshfeld surface analysis was performed and the associated two-dimensional fingerprint plots were obtained with 4.et al., 2016S)-5-chloro-N-({2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]oxazolidin-5-yl}methyl)-thiophene-2-carboxamide [(I): Shen et al., 2018et al., 2010et al., 2010et al., 2012R)-2-phenoxy-1-ethanone [(V): Caracelli et al., 2011A search of the Cambridge Structural Database , adjacent molecules are connected via O—H⋯O and C—H⋯O hydrogen bonds and C—H⋯π interactions into a zigzag chain along the b-axis direction. In (V), molecules are linked into supramolecular arrays two molecules thick in the bc plane through C—H⋯O, C—H⋯S and C—H⋯π interactions.In the crystal of (I)5.The title compound was synthesized using our recently reported procedure  = 1.2 or 1.5Ueq(C). The hydroxyl H atom was found in a difference-Fourier map and was refined freely.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022005928/tx2051sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022005928/tx2051Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022005928/tx2051Isup3.cmlSupporting information file. DOI: 2176709CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The first crystal structure is reported of a molecule containing the 1,4,2-dithiazole-5-thione heterocycle. The packing features aromatic π–π stacking, weak C—H⋯S interactions and short S⋯S interactions. 8H5NS3, the dihedral angle between the heterocyclic ring and the phenyl ring is 2.62 (5)°. In the extended structure, aromatic π–π stacking between the 1,4,2-dithiazole-5-thione moiety and the phenyl ring is observed [centroid–centroid distances = 3.717 (6) and 3.712 (6) Å]. The almost planar molecules arrange themselves in parallel chains of head-to-tail molecules oriented by a network of weak C—H⋯S contacts close to the sum of their van der Waals radii within the chains. All the hydrogen atoms participate in hydrogen-bonding interactions with the sulfur and nitrogen atoms of adjacent molecules. C=S⋯S contacts between the chains that are significantly shorter than the sum of their van der Waals radii also impact the overall packing.In the title compound, C This pattern of bond lengths is in agreement with a more extensive, and less localized, π delocalization in this heterocycle than in the comparable oxathiazolone derivatives.Within the heterocycle moiety, the molecule shows significant  °] on the a axis. Head-to-toe hydrogen-bonding interactions between C5 and C6 donors to the exocyclic thione S3 with H⋯S distances of 2.96 and 3.11 Å, respectively , H5⋯S1 (3.04 Å), H7⋯S3 (3.06 Å) and H8⋯S2 (3.10 Å). The sulfur–sulfur interactions occur as a chain out of plane between the thione S3 and S1 atoms within the ring  = 0.671; UV–visible (CH2Cl2) λmax nm (log ɛ): 256 (4.29), 361 (4.20), 1H NMR 60 MHz, CDCl3) δ = 7.53 ppm (multiplet).A solution of trichloromethanesulfenyl chloride  in chloroform (10.17 g) was added dropwise to a warmed solution of thiobenzamide  in chloroform (240 ml) according to a literature procedure (Greig 6.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902200888X/hb8037sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902200888X/hb8037Isup2.hklStructure factors: contains datablock(s) I. DOI: 2205416CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecular structure features an angular (phenyliodosyl)benzene cation, the geometry of which was hitherto undescribed in the literature: in the cation, both I—C bonds are approximately normal to the I=O bond, forming a C—I—C angle of 95.36 (4)°. The crystal structure displays O—H⋯O, O—H⋯I and O—H⋯S hydrogen bonding.The structure of the title salt (systematic name: oxodiphenyl-λ This iodosyl salt arose from the reaction of iodoxybenzene with sodium hydroxide benzene tosylate dihydrate °. The bond lengths of C1—I1 and C7—I1 are 2.1289 (11) and 2.1370 (12) Å, respectively. These values are comparable to the sum of the van der Waals radii -one, i.e. the Dess–Martin periodinane, with a C—I bond length between the phenyl and iodine of 2.1025 (16) Å  Å], resides nearly perpendicular at 77.55 (4)° to the I1—O1 bond. This bond length is shorter than the sum (3.05 Å) of the covalent radii, and this is analogous to secondary bonding that was observed by Rentzeperis in his bis(diphenyliodonium I-oxide) diacetate trihydrate, between the acetate anion and the iodonium I-oxide centre  diacetate trihydrate  and 2.8118 (10) Å, respectively.Zhdankin synthesized a tosylate derivative of 2-iodoxybenzoic acid. Crystals of the final product could not be isolated, but the intermediate mixed tosylate-acetate derivative was analysed. The I—O bond lengths in this iodine(V) compound had distances of 2.080 (2), 2.213 (2), 2.027 (2) and 1.998 (2) Å (Yusubov I-oxide) diacetate trihydrate also adopted a distorted octahedral geometry, albeit via a dimeric coordinating structure °, the C7—I1—O3 angle of 176.33 (4)°, the O1—I1—O4 angle of 77.55 (4)° with the coordinating tosylate anion and the C1—I1—C7 angle of 95.36 (4)° complete the distorted octahedral geometry. The accompanying tosylate anion and water molecules occupy apical and equatorial positions to stabilize the monomeric complex. Bis and 1.99 (2) Å, respectively, as viewed down the a axis  diacetate trihydrate  was added to the cooled filtrate and a white precipitate formed. The suspension was stirred for an additional 30 min and then filtered. The compound was washed with a minimal amount of diethyl ether (10 ml) and ice-cold water (10 ml). The product  matched known 1H, 13C and FTIR data benzene tosylate dihydrate was synthesized according to a modified procedure by Chen 2007 and is iCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314622005223/bh4069sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314622005223/bh4069Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622005223/bh4069Isup3.cmlSupporting information file. DOI: 2173122CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


At 293 K, [tBuNHC2Me][Br]2·H2O crystallizes in the P21/c space group, while [tBuNHC2Et][Br]2·2H2O crystallizes in the P21/n space group at 100 K. At 112 K, [MesNHC2Me][Br]2·2H2O crystallizes in the orthorhombic space group Pccn while [MesNHC2Et][Br]2·4H2O crystallizes in the P21/c space group at 100 K.The crystal structures of ligand precursor bis(imidaozolium) salts 1,1′-methylenebis(3- tert-butylimidazolium) dibromide monohydrate, C15H26N4+·2Br−·H2O or [tBuNHC2Me][Br]2·H2O, 1,1′-bis(3-tert-butylimidazolium) dibromide dihydrate, C16H28N4+·2Br−·2H2O or [tBuNHC2Et][Br]2·2H2O, 1,1′-methylenebis[3-imidazolium] dibromide dihydrate, C25H30N42+·2Br−·2H2O or [MesNHC2Me][Br]2·2H2O, and 1,1′-bis[3-imidazolium] dibromide tetrahydrate, C26H32N42+·2Br−·4H2O or [MesNHC2Et][Br]2·4H2O, are reported. At 293 K, [tBuNHC2Me][Br]2·H2O crystallizes in the P21/c space group, while [tBuNHC2Et][Br]2·2H2O crystallizes in the P21/n space group at 100 K. At 112 K, [MesNHC2Me][Br]2·2H2O crystallizes in the orthorhombic space group Pccn while [MesNHC2Et][Br]2·4H2O crystallizes in the P21/c space group at 100 K. Bond distances and angles within the imidazolium rings are generally comparable among the four structures. All four bis(imidazolium) salts co-crystallize with one to four molecules of water.The crystal structures of ligand precursor bis(imidazolium) salts 1,1′-methylenebis(3- Bis(imidazolium) salts, [RNHC2R1][X]2 are relatively modular in that modifications can be relatively easily made to exterior groups attached to each NHC (R), the moiety linking the two NHC groups (R1), and the counter-ion (X). One general synthetic approach for synthesizing bis(imidazolium) salts is where two equivalents of an alkyl or aryl imidazole are combined with one equivalent of an organic dihalide reagent and refluxed to afford the final product  salts can be relatively easily synthesized from alkyl or aryl imidazoles, and some are also commercially available.Bis(imidazolium) salts are common precursors for the synthesis of bidentate tert-butyl (tBu) and mesityl (Mes) exterior R groups and methylene (Me) or ethylene (Et) linking R1 groups. [MesNHC2Et][Br]2 was even reported to be a stand-alone catalyst for the conversion of arylaldehydes to carboxylic acids in combination with water and K2CO3 in DMSO  were reported to be more effective for hydrosilylation reactions with RhI complexes than ethylene linkers  salts are those with tBuNHC2Me][Br]2 and [tBuNHC2Et][Br]2 were used as precursors for synthesis of rhodium complexes 2-assisted deprotometalation  salts with tert-butyl and mesityl ancillary groups.While bis(imidazolium) salts are common ligand precursors, few have been structurally characterized  salts were recrystallized from hot methanol and each compound co-crystallizes with one or more molecules of water. Fig. 1tBuNHC2Me][Br]2·H2O and [tBuNHC2Et][Br]2·2H2O are mostly the same within experimental error, with backbone C2—C3 distances of 1.348 (4) and 1.349 (3) Å, respectively. The N—C distances are also mostly comparable with [tBuNHC2Me][Br]2·H2O having an N1—C2 and an N2—C3 distance of 1.389 (3) Å and N1—C1 and N2—C1 distances both being 1.337 (3) Å, while [tBuNHC2Et][Br]2·2H2O has an N1—C2 distance of 1.388 (3) Å, an N2—C3 distance of 1.384 (3) Å, an N1—C1 distance of 1.327 (3) Å and an N2—C1 distance of 1.331 (3) Å. For the linker, the N2—C7 distance is 1.463 (3) Å for [tBuNHC2Me][Br]2·H2O and 1.468 (3) Å for [tBuNHC2Et][Br]2·2H2O.Bond distances in the imidazolium rings of [tBuNHC2Me][Br]2·H2O and [tBuNHC2Et][Br]2·2H2O. For [tBuNHC2Me][Br]2·H2O, bond angles include C1—N1—C2 at 108.2 (2)°, N1—C2—C3 at 107.6 (2)°, C2—C3—N2 at 106.9 (2)°, C3—N2—C1 at 108.6 (2)°, and N2—C1—N1 at 108.7 (2)°. For [tBuNHC2Et][Br]2·H2O, bond angles include C1—N1—C2 at 108.21 (19)°, N1—C2—C3 at 107.3 (2)°, C2—C3—N2 at 106.9 (2)°, C3—N2—C1 at 108.54 (19)°, and N2—C1—N1 at 109.02 (19)°.Bond angles in the imidazolium rings are also quite similar in [MesNHC2Me][Br]2·2H2O while Fig. 4MesNHC2Et][Br]2·4H2O. Notably, [MesNHC2Et][Br]2·4H2O is the only compound of the four for which the asymmetric unit contains only half of the molecule.Fig. 3MesNHC2Me][Br]2·2H2O and [MesNHC2Et][Br]2·4H2O are mostly the same within experimental error, with backbone C2—C3 distances of 1.344 (3) and 1.3506 (19) Å, respectively. N—C distances are also mostly the same with [MesNHC2Me][Br]2·2H2O having an N1—C2 distance of 1.387 (3) Å, an N2—C3 distance of 1.380 (3) Å, an N1—C1 distance of 1.326 (3) Å, and an N2—C1 distance of 1.341 (3) Å. Similarly, [MesNHC2Et][Br]2·4H2O has an N1—C2 distance of 1.3872 (16) Å, an N2—C3 distance of 1.3841 (16) Å, an N1—C1 distance of 1.3322 (16) Å and an N2—C1 distance of 1.3314 (16) Å. For the linker, the N2—C7 distance is 1.457 (3) Å for [MesNHC2Me][Br]2·2H2O and 1.4653 (16) Å for [MesNHC2Et][Br]2·4H2O.Bond distances in the imidazolium rings of [MesNHC2Me][Br]2·2H2O and [MesNHC2Et][Br]2·4H2O. For [MesNHC2Me][Br]2·2H2O, bond angles include C1—N1—C2 at 108.92 (17)°, N1—C2—C3 at 107.20 (19)°, C2—C3—N2 at 106.95 (19)°, C3—N2—C1 at 108.96 (17)°, and N2—C1—N1 at 107.96 (18)°. For [MesNHC2Et][Br]2·4H2O, bond angles include C1—N1—C2 at 108.51 (11)°, N1—C2—C3 at 107.19 (11)°, C2—C3—N2 at 106.87 (11)°, C3—N2—C1 at 108.89 (11)°, and N2—C1—N1 at 108.54 (11)°. Overall, these data support that changing the linker R1 group from methylene to ethylene does not significantly affect the imidazolium ring structures.Bond angles in the imidazolium rings are also mostly the same for [3.tBuNHC2Me][Br]2·H2O is stabilized by hydrogen bonding  Å. One tert-butyl group has positional disorder.The supramolecular structure of [ng Fig. 5. Distancng Fig. 5. HydrogetBuNHC2Et][Br]2·2H2O is stabilized by extensive hydrogen bonding  and Br1⋯H1A (2.398 Å).The supramolecular structure of [ng Fig. 6. Distancng Fig. 6. SeveralMesNHC2Me][Br]2·2H2O is also stabilized by hydrogen bonding  Å, O2⋯H1E at 2.001 (3) Å, and Br1⋯H1D at 2.585 (2) Å.The supramolecular structure of [Br]2·H2O, [tBuNHC2Et][Br]2·2H2O, or [MesNHC2Et][Br]2·4H2O. A deposited dataset for [MesNHC2Me][Br]2·2H2O was found . The procedure was adapted from a literature procedure , approximately 100 mL of methanol, and approximately 25 mL of deionized water and a stir bar, then heated to 343 K under reflux. 7.81 mL of 37% formaldehyde  were added, followed by 3.70 mL of ammonium hydroxide  added dropwise over 5 minutes while stirring. The solution was refluxed at 343 K for 5 h, resulting in a light red–orange solution. Excess solvent was removed in vacuo, and the resulting product was diluted with approximately 150 mL of dichloromethane and washed twice with 50 mL of deionized H2O until the aqueous layers ran clear. The product was vacuum distilled at ∼373 K, yielding a clear liquid, which was weighed in a tared vial, resulting in 7.95 g (34% yield) of tBuIm, and characterized by 1H NMR spectroscopy in CDCl3.Synthesis of 1--1HMesIm).-imidazole,  of tan crystals, which were characterized by 1H NMR spectroscopy and identified as MesIm.Synthesis of 1,1′-di(tert-butyl)-3,3′-methylene-diimidazolium dibromide, [tBuNHC2Me][Br]2. 1.850 g  of tbuIm and 0.4194 mL  of dibromomethane, a stir bar, and ∼20 mL of toluene were stirred in a 50 mL round-bottomed flask. The solution was then heated to 423 K and refluxed for 46 h, resulting in the formation of a dark orange–brown solution. The solution was cooled in an ice bath, resulting in a fine white precipitate which was collected via vacuum filtration, washed twice with ∼5 mL of cold toluene, filtered and dried. 1.120 g (78.02% yield) of a fine white solid identified as [tBuNHC2Me][Br]2 were isolated. Crystals suitable for X-ray diffraction were obtained by recrystallization from hot methanol. The product was characterized by 1H NMR spectroscopy. The 1H NMR data were consistent with those previously reported -3,3′-ethylene-diimidazolium dibromide [tBuNHC2Et][Br]2. A 250 mL round-bottomed flask was charged with 2.017 g  of tbuIm, 0.562 mL  of dibromoethane, a stir bar, and ∼20 mL of toluene. The mixture was refluxed at 423 K and stirred for 46 h, at which point the solution was a rusty brown color. The flask was then placed in an ice bath, and the resulting precipitate was collected via vacuum filtration and washed twice with ∼5 mL of cold toluene. The resulting solids were dried and weighed, yielding 1.727 g (61% yield) of [tBuNHC2Et][Br]2 and single crystals suitable for X-ray diffraction were obtained via recrystallization from hot methanol. 1H NMR data were consistent with those previously reported -3,3′-methylene-diimidazolium dibromide, [Br]2 suitable for X-ray diffraction and characterized by 1H NMR.MesNHC2Et][Br]2.Synthesis of 1,1′-di(mesityl)-3,3′-ethylene-diimidazolium dibromide, [Br]2.6.SHELXL and included as riding contributions with distances of 0.95 Å for C—H, 0.99 Å for CH2 and 0.98 Å for CH3. Methyl H atoms were allowed to rotate but not to tip to best fit the experimental electron density. Uiso values of riding H atoms were set to 1.2 times Ueq(C) for CH and CH2, and 1.5 times Ueq(C) for CH3 and H2O. For [tBuNHC2Me][Br]2, the SADI command of SHELX was used to model disorder in one of the tert-butyl moieties for N4—C0AA and N4—C12, C0AA—C00N and C14—C12, and C1AA—C0AA and C13—C12 to restrain distances within a sigma of 0.02 Å. The population parameters for the disordered tert-butyl groups are 0.54019 for C12–C14, and 0.45981 for C00N, C0AA, and C1AA. The highest peak and deepest hole are both near a heavy atom Br1 with a distance of 0.88 Å from the highest peak of 1.49 e Å−3 and a distance of 0.73 Å from the deepest hole of −1.10 e Å−3.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022008003/mw2188sup1.cifCrystal structure: contains datablock(s) sces01006_0m, est01043_0m, at01019_0ma, est01041d_0ma, global. DOI: 10.1107/S2056989022008003/mw2188sces01006_0msup2.hklStructure factors: contains datablock(s) sces01006_0m. DOI: 10.1107/S2056989022008003/mw2188est01043_0msup3.hklStructure factors: contains datablock(s) est01043_0m. DOI: 10.1107/S2056989022008003/mw2188at01019_0masup4.hklStructure factors: contains datablock(s) at01019_0ma. DOI: 10.1107/S2056989022008003/mw2188est01041d_0masup5.hklStructure factors: contains datablock(s) est01041d_0ma. DOI: Click here for additional data file.10.1107/S2056989022008003/mw2188sces01006_0msup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989022008003/mw2188est01043_0msup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989022008003/mw2188at01019_0masup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989022008003/mw2188est01041d_0masup9.cmlSupporting information file. DOI: 2195736, 2195735, 2195734, 2195733CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: