N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide   •GalNAc-T glycosyltransferase bump-and-hole engineering retains structure and substrate specificity•Bumped UDP-GalNAc analogs can be delivered to the living cell•Cellular substrates of individual GalNAc-Ts can be bioorthogonally tagged•Glycoproteomics identifies GalNAc-T-specific glycosylation sites Schumann et al. establish a cellular glycosyltransferase bump-and-hole system to chemically tag the substrates of individual isoenzymes of the GalNAc-T family. Glycans are the prime example for this; the human glycome is constructed by the combinatorial activity of more than 250 glycosyltransferases (GTs) with both hierarchical and competing activities. On the cell surface, glycans play a central role in modulating signal transduction, cell-cell interactions, and biophysical properties of the plasma membrane  transferase family . Transferring GalNAc to Ser/Thr side chains, GalNAc-Ts initiate abundant O-linked glycosylation in the secretory pathway  at 1.8-Å resolution. Comparison of BH-T2 and WT-T2 (PDB: 2FFU) revealed complete conservation of both the three-dimensional enzyme architecture and bound ligand structure (α distances), respectively, likely to account for the changes elsewhere in the active site. BH-T2 thus retains the native structural properties of the WT-GalNAc-T enzyme.As bump-and-hole engineering of a GT family in living cells has no precedent, we first set out to understand the structural implications of this approach. All GalNAc-Ts are type II transmembrane proteins with luminal GT and lectin domains connected through a flexible linker . We crystructure . In BH-Ttructure . The muttructure B. Two gl2+, and UDP-GalNAc analog 1 at 3.1-Å resolution helped us visualize how the BH-T2 active site mutations affect enzyme-substrate binding. In comparison with a corresponding WT-T2/UDP-GalNAc/Mn2+/EA2 complex (PDB: 4D0T), UDP-sugar binding is completely conserved (1 (M of BH-T2 toward 1 (2.6 μM) is approximately 10-fold lower than the KM of WT-T2 toward UDP-GalNAc (30 μM) (cat of BH-T2/1 (0.158 s−1) is approximately 5-fold lower than the kcat of WT-T2/UDP-GalNAc (0.813 s−1) indicates that these additional interactions may slightly slow the ring distortion that is needed to mediate the nucleophilic attack  . In turnc attack .in vitro glycosylation experiments and analyzed by self-assembled monolayers for matrix-assisted desorption/ionization MS  epitope tag exhibited doxycycline (Dox) dose-dependent expression under the control of a tetracycline-responsive promoter in stably transfected HepG2 cells . All tesalNAc-Ts . These ralNAc-Ts E. Import;1, 2, and 3 were not biosynthesized from their sugar-1-phosphate precursors in the living cell  analogs has previously prompted the engineering of AGX1 to recognize the corresponding GlcNAc-1-phosphate analog as a substrate  of cell lysates. Gly and Ala mutants of Phe383 efficiently biosynthesized bumped UDP-GalNAc analog 1, while neither Phe381 single mutants nor any Phe381/Phe383 double mutants produced 1 despite equal expression levels  was biosynthesized by engineered AGX1F383A, hereby called mut-AGX1 .In HPAEC-PAD chromatograms of cells biosynthesizing UDP-GalNAc analog analog 8 A: first,ite peak C–S3E. In1 in hand, we set out to probe isoenzyme-dependent glycosylation in the living cell. We cloned FLAG-tagged WT- or mut-AGX1 under a constitutive promoter into an expression vector containing VSV-G-tagged WT or BH versions of GalNAc-T1 or T2 under the control of a Dox-inducible promoter  complex  were present . We found all these proteins enriched  from lysates of cells carrying functional T1 or T2 bump-and-hole systems  over control cells lacking any component  as an indicator for a bumped glycan to trigger peptide sequencing by electron-transfer dissociation : previous data showed that upon T1-KO, glycosylation of Thr221, Thr226, and Ser228 is slightly decreased, while upon T2-KO, all three sites had increased occupancy .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Carolyn R. Bertozzi (schjoldager@sund.ku.dk) or Hans H. Wandall (hhw@sund.ku.dk) should be contacted. All other cell lines generated in this study are available through the Lead Author. All plasmids generated in this study are available through the Lead Author; of these, plasmids from addgene are protected by an UBMTA and can be obtained for non-commercial use under an equal UBMTA. Newly synthesized chemicals will be shared while stocks last, and synthetic procedures are in the Supporting Information.HepG2-T1-KO, HepG2-T2-KO cells and all cell lines derived from these are subject to an MTA with the University of Copenhagen, and Katrine T. Schjoldager (6E7I and PDB: 6NQT). The mass spectrometry proteomics and glycoprotemics data have been deposited to the ProteomeXchange Consortium via the PRIDE  were a gift from Michael Bassik  and propagated in RPMI (Thermo Fisher) with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C with 5% CO2. Expression of spCas9 was confirmed by western blot. K-562 GALE-KO cells were derived from K-562-spCas9 cells, and were grown under the same conditions, but with addition of 200 μM GalNAc and 20 μM galactose to the culture medium. GALE-KO cells were maintained in standard growth medium supplemented with 20 μM galactose and 200 μM GalNAc. GALE was not detectable by western blot using mouse anti-GALE sc-390407  over at least 20 passages. Cell authentication was performed via STR profiling and species authentication. Female K-562 cells were a gift from Jonathan Weissman . Cells were grown in RPMI with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL). Cell authentication was performed via STR profiling and species authentication. HEK293T (ATCC CRL-3216) of unknown sex were from ATCC and grown in DMEM (Thermo Fisher) with 10% (v/v) FBS (Thermo Fisher), penicillin (100 U/mL) and streptomycin . Cell authentication was performed via STR profiling and species authentication. Freestyle™ 293-F cells of unknown sex were propagated in FreeStyle™ 293 Expression Medium (Thermo Fisher) with 10% (v/v) fetal bovine serum  at 37 °C and 8% CO2 with orbital rotation at 135 rpm. Cells were not authenticated. HepG2 cells (ATCC HB-8065), HepG2-T1-KO and HepG2-T2-KO cells  were propagated in low-glucose DMEM  with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were not authenticated.All cell lines were mycoplasma-free, as confirmed using the MycoAlert Kit . Female K-562 cells with stable expression of The following nomenclature of GalNAc-T and AGX1 genes will be used herein: WT-T1, WT-T2: wild-type constructs; BH-T1: I238A/L295A; BH-T2: I253A/L310A; mut-AGX1: F383A.Primers and gBlocks were from Integrated DNA Technologies  and Elim Biopharm . Restriction enzymes were from New England Biolabs except for PasI . PfuUltra II  and Advantage Genomic LA polymerase  were used for T1 and T2, respectively. T4 DNA ligase  and LONG DNA ligase (Takara) were used for ligation of T1 and T2, respectively. Q5 high-fidelity DNA polymerase (New England Biolabs) was used for overlap extension PCR. All plasmids were sequenced before use at Elim Biopharmaceuticals, Inc. .The plasmids pSBtet-GH and pSBbi-GH were a gift from Eric Kowarz  .Full length human GalNAc-T2 (EBI accession number LC043140.1) in pCMV-NTAP was a kind gift from Lawrence Tabak  . T2 was NM_020474) was used as a gBlock (IDT) with overhangs and NotI/BamHI restriction sites, and amplified with the primers T1_F1 and T1_R1 prior to cloning into pFLAG-CMV-5.1 using a NotI/BamHI strategy. Site-directed mutagenesis (I238A/L295A) was performed at this step according to a published procedure  and medium was renewed (30 mL). Cells were harvested and conditioned supernatant collected after one, three, and five days, and medium was renewed each time. Conditioned supernatant was passed through a freshly-packed column containing 2 mL HisPur NiNTA resin slurry (Thermo Fisher) pre-conditioned with water and Wash Buffer . The resin was treated with Wash Buffer (2x15 mL), and subsequently with Elution Buffer ). Eluted protein was dialyzed using Amicon Ultra-15 centrifuge filters  against Crystallization Buffer . Protein was stable for one week at 4 °C. For long-term storage at −80 °C, 80% (v/v) aq. glycerol was added to a final concentration of 25% (v/v). Protein was re-buffered to Crystallization Buffer immediately prior to use. Typically, 1.2 mg protein was purified from one 30 mL sample of conditioned supernatant.The plasmid pOPING was a gift from Ray Owens  . A secrec-CMV-19  was clonCrystals were grown using sitting drop vapor diffusion at room temperature. Drops were set-up using a Douglas Oryx8 Nanodrop Dispensing Robot .2+ and EA2 peptide were obtained through co-crystallization using 0.15 μL of 5 mM MnCl2, 5 mM UDP, 6 mM EA2, and 10 mg/mL protein in Crystallization Buffer and 0.15 μL precipitant solution (11%–32% (v/v) PEG8000, 0.1 mM HEPES, pH 6.5-8.5) against 80 μL of precipitant solution. Crystals were grown for 1 week before being cryoprotected in 16% ethylene glycol and frozen in liquid nitrogen before diffraction.Crystals of BH-T2 complexed with UDP, Mn1 and Mn2+ were obtained through soaking of EA2 and 1 into co-crystals of BH-T2 with UDP and Mn2+. EA2 peptide electron density was too weak to properly discern in crystal structures. Initial drops were created using 0.15 μL of 5 mM MnCl2, 5 mM UDP, 10 mg/mL T2 BH protein in 25 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1 mM TCEP and 0.15 μL precipitant solution  against 80 μL of precipitant solution. Crystals were grown for 18 days before soaking and diffraction. Crystals were soaked in a solution with EA2 by adding 0.45 μL of a 50 mM EA2 solution in water to the original drop for 30 minutes to 2 hours. Crystals were harvested and soaking was performed with a solution of 20 mM 1 with 16% ethylene glycol, 11%–23% PEG8000, 0.1 mM HEPES, 5 mM MnCl2, pH 7.5-8.5 for 1-2 hours.Crystals of BH-T2 complexed with 2+ (PDB 6E7I) was performed at 95K using ALS Beamline 5.0.1  and single wavelength of 0.97741 Å, with a Dectris Pilatus3 S 6M Detector .Single crystal X-ray diffraction of BH-T2/EA2/UDP/Mn1/Mn2+ (PDB 6NQT) was performed at 100K using Stanford Synchrotron Radiation Lightsource Beamline 7-1  and single wavelength of 0.9753 Å, with an ADSC Quantum 315r CCD Detector .Single crystal X-ray diffraction of BH-T2/2FFU for PDB: 67EI and PDB: 4D0T for PDB: 6NQT) as the templates , at 37 °C for 3 h  using the primers AGX1 subcloning_F/_R, purified by gel extraction, and subjected to an overlap extension PCR with equimolar amounts (10 fmol each) of the gBlocks (IDT) AGX1_gBlock_5′ and AGX1_gBlock_3′ using 10 mM dNTPs and Q5 polymerase. Briefly, a 50 μL reaction was run for 15 cycles using the following protocol: 95 °C (2 min), 95 °C (30 s), 64 °C (30 s), 72 °C (30 s – return to step 2), 72 °C (5 min), then treated with the primers (500 nmol) AGX1 extension_F and AGX1 extension_R and continued for another 18 cycles with an annealing temperature of 55 °C. The resulting PCR product was used to replace the eGFP cloning sequence of pSBtet-GH containing T1 or T2 using an XcmI/PasI strategy to digest the vector and In-Fusion HD Cloning Kit to insert AGX1 sequences. AGX1-containing pSBtet vectors are named pSBtet-WT-hAGX1 or pSBtet-mut-hAGX1. For constitutive expression of GalNAc-Ts, eGFP coding sequence was accordingly replaced with AGX1 in pSBbi-GH by GeneArt (Thermo).Vectors herein are pSBtet-GH , pSBtet-WT-hAGX , pSBtet-mut-hAGX1 , pSBtet-GH-WT-T1 , pSBtet-GH-BH-T1 , pSBtet-GH-WT-T2 , pSBtet-GH-BH-T2 , pSBtet-mut-hAGX1-WT-T1 , pSBtet-WT-hAGX1-BH-T1 , pSBtet-mut-hAGX1-BH-T1 , pSBtet-mut-hAGX1-WT-T2 , pSBtet-WT-hAGX1-BH-T2 , pSBtet-mut-hAGX1-BH-T2 , pSBbi-WT-hAGX1-BH-T1 , pSBbi-mut-hAGX1-WT-T1 , pSBbi-mut-hAGX1-BH-T1 , pSBbi-WT-hAGX1-BH-T2 , pSBbi-mut-hAGX1-WT-T2 , pSBbi-mut-hAGX1-BH-T2 .GALE were chosen according to in silico (www.synthego.com). SgRNAs were cloned into the vector pU6-sgGAL4-4 (Single-guide (sg) RNAs GALE_sgRNA1 and GALE_sgRNA2 targeting human sgGAL4-4 , a gift HEK293T cells were seeded at 1.2 million cells per well of a 6-well plate. After 24 h, cells were transfected with 1.5 μg sgRNA-coding vector and a third generation lentiviral packaging mix  and 4 μg/mL of AlexaFluor488 conjugated streptavidin (Thermo Fisher) on ice for 30 min. The cells were washed twice with PBS + 0.5% BSA and viable cells were sorted based on AlexaFluor488 fluorescence intensity and low SYTOX Red (Thermo) staining according to a published procedure  were added. After another 24 h, cells were harvested and treated with fresh growth medium. After another 24 h, cells were harvested and treated with growth medium containing 1.25 μg/mL puromycin. Selection was carried out for 96 h, and cells were expanded in puromycin-free growth medium. The knockout cells were fed 100 uM of GalNAc for 2 days before FACS sorting. After 48 h, the cells were stained with 5 μg/mL biotinylated rocedure . UDP-Galrocedure C. Both sTet-system approved FBS (Takara) was used to propagate all cell lines transfected with pSBtet-GH-based plasmids. pCMV(CAT)T7-SB100 was a gift from Zsuzsanna Izsvak  .HEK293T cells were transfected with pIRES-puro3 plasmids containing AGX1 constructs using TransIT-293  according to the manufacturer’s instructions and 37.5 μg DNA per 15 cm dish or 15 μg DNA per 10 cm dish. After 24 h, medium was aspirated, and cells were either treated with fresh growth medium and compounds for analysis of nucleotide-sugar biosynthesis (see below), or growth medium containing 5 μg/mL puromycin (Sigma Aldrich) for selection of stable cells for two weeks.K-562 cells were transfected with pSBtet-based plasmids using Lipofectamine LTX (Thermo Fisher) according to the manufacturer’s specifications, with 200000 cells in 1 mL growth medium, 1.25 μg pSBtet and 62.5 ng pCMV(CAT)T7-SB100 plasmid DNA. After 24 h, cells were harvested and selected in growth medium containing 150 μg/mL hygromycin B (Thermo Fisher) for 7-10 days to obtain stable cells.HepG2, HepG2-T1-KO and HepG2-T2-KO cells were transfected with Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s instructions, e.g., using 2.5 μg pSBtet and 125 ng pCMV(CAT)T7-SB100 plasmid DNA per well of a 6-well plate. After 24 h, medium was aspirated, and cells were treated with fresh growth medium containing 600 μg/mL (HepG2) or 500 μg/mL (HepG2-T1-KO and HepG2-T2-KO) hygromycin B for two weeks to obtain stable cells. Following selection, cells were propagated in 200 μg/mL hygromycin B in growth medium.HepG2 cells stably transfected with pSBtet-GH, pSBtet-GH-WT-T1, pSBtet-GH-BH-T1, pSBtet-GH-WT-T2, or pSBtet-GH-BH-T2 were seeded into 6-well plates at 40% confluency. After 24 h, the supernatant was aspirated and changed to fresh growth medium without hygromycin B, containing the indicated concentration of doxycycline . After 24 h, the treatment was repeated in fresh growth medium. After another 24 h, cells were washed with PBS (1 mL), treated with ice-cold PBS containing 1 mM EDTA (1 mL) and incubated for 10 min at 4 °C. Cells were transferred to a 1.5 mL reaction tube, harvested , and resuspended in ice-cold 50 mM Tris-HCl pH 8, 5 mM EDTA, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS (0.2 mL) containing cOmplete protease inhibitors . Cells were briefly vortexed, incubated for 30 min at 4 °C with agitation, and centrifuged . The supernatant was transferred to a new tube, and the protein concentration was measured by BCA (Thermo Fisher). SDS-PAGE and western blot were performed using 15 μg protein per lane, and blots were decorated with mouse anti-VSV-G-tag P5D4  and rabbit anti-GAPDH-HRP conjugate ab185059 (abcam).Membrane protein glycosylation was performed on a membrane fraction from untransfected or stable HepG2 cells. Cells were seeded in a 15 cm dish, grown to 50% confluency and treated with doxycycline . After 24 h, doxycycline treatment was repeated. After another 24 h, cells were washed with PBS (10 mL) and scraped in PBS (5 mL). Cells were harvested by centrifugation , washed once with ice-cold PBS (2 mL) and frozen at −80 °C. Cells were thawed on ice and fractionated using the Subcellular Fractionation Kit for Cultured Cells (Thermo Fisher) according to the manufacturer’s instructions, with 0.4 mL of both Cytoplasmic Extraction Buffer and Membrane Extraction Buffer. Protein concentration was measured by BCA (Thermo Fisher), and fractions were frozen after addition of 80% (v/v) glycerol .In vitro glycosylation reactions were performed using 10 μg membrane protein of stably transfected HepG2 cell lines in 20 μL reaction volume containing 62.5 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MnCl2, 250 μM UDP-GalNAc analog and 500 μM UDP-GalNAc at 37 °C for 12 h. Reactions were heat-inactivated at 95 °C for 20 s and cooled to 4 °C. Then, alkyne-containing reaction mixtures were sequentially treated with equal volumes (1.25 μL each) of 2 mM biotin-PEG4-azide (Thermo Fisher), 2 mM BTTAA , 20 mM CuSO4 and 100 mM sodium ascorbate . Click reactions were carried out at room temperature for 2 h and quenched by addition of 50 mM EDTA. Reaction mixtures were then subjected to SDS-PAGE and blotted on nitrocellulose membranes. The total protein amount was assessed using the REVERT protein staining kit , and biotinylation was detected using IRDye 800CW Streptavidin (LI-COR Biosciences) according to the manufacturer’s instructions.In vitro glycosylations were replicated using a membrane protein fraction from untransfected HepG2 cells that was depleted for internal GalNAc-T activity by three heat (90 °C)-cool (4 °C) cycles of 30 s each. Soluble FLAG-tagged WT- or BH-GalNAc-Ts (8) were added at a final concentration of 20 nM (T1) or 10 nM (T2), along with bumped UDP-GalNAc analog (250 μM) and UDP-GalNAc (500 μM). Glycosylation, click reaction and Streptavidin detection were performed as described above.2+ and 100 mg/mL Mg2+ (DPBS), and fixed with 4% (v/v) paraformaldehyde (Sigma Aldrich) in PBS for 10 min in the dark. Cells were washed with 50 mM ammonium chloride in DPBS and DPBS with 2% (w/v) BSA (Incubation Buffer). Cells were then permeabilized with 0.5% (v/v) Tween-20 in incubation buffer for 10 min in the dark, and subsequently treated with antibody solutions in the same buffer: mouse anti-VSV-G-tag, (1:200), and rabbit anti-Giantin . Cells were incubated for 1 h, washed twice with Incubation Buffer with 0.1% Tween-20, and incubated with secondary antibodies in Incubation Buffer containing 0.5% (v/v) Tween-20: Donkey anti-mouse Alexa Fluor 568  and donkey anti-rabbit Alexa Fluor 647 . Cells were incubated for 1 h in the dark, washed twice with Incubation Buffer with 0.1% (v/v) tween-20, and mounted on glass slides using ProLong Diamond Antifade mounting solution (Thermo Fisher). Cells were imaged using a Nikon A1R+ Resonant Scanning Confocal Microscope.Fluorescence microscopy was performed with stably transfected HepG2 cells. Cells were Dox-induced  for 24 h, then trypsinated and transferred in growth medium to a 24 well plate containing circular 12 mm coverslips  pre-treated with human fibronectin  for 20 min and washed with PBS. Cells were treated with Dox again and incubated for another 24 h. Supernatant was aspirated, cells were washed in PBS with 100 mg/L CaCells expressing AGX1-FLAG (transient or stably transfected HEK293T in 20 mL growth medium in a 15 cm dish or 5 million stable K-562 in 4 mL growth medium) were treated with caged GalNAc-1-phosphate analogs  or DMSO vehicle. After 7 h, K-562 cells were harvested  and the supernatant was aspirated. HEK293T cells were washed once on the plate with cold PBS (8 mL), scraped in cold 1 mM EDTA in PBS (8 mL), transferred to a conical tube and harvested . Cell pellets were resuspended in PBS (1 mL), 0.8 mL of that suspension was transferred to O-ring tubes  and harvested. Zirconia/silica beads  were added at a similar volume as the cell pellet, followed by 1:1 acetonitrile/water (1 mL). Cells were lysed with a bead beater  for 30 s at 6 m/s, and the mixture was left at 4 °C for 10 min. Samples were centrifuged , and the resulting supernatant was transferred to a new 1.5 mL tube. The solvent was removed by speed vac. The residue was resuspended in a solution of 15 μM ADP-α-D-glucose (Sigma Aldrich) in LCMS-grade water . The solution was membrane-filtered  using a 3 kDa cut-off filter  and analyzed by high performance anion exchange chromatography. The residual cell suspension in PBS (0.2 mL) was harvested, and the pellet was resuspended in M-PER lysis buffer (Thermo Fisher) containing cOmplete protease inhibitor (0.2 mL). The solution was incubated for 10 min at room temperature and centrifuged . The protein concentration of the supernatant was measured by BCA, and samples were used for analysis of protein expression.High performance anion exchange chromatography was performed on an ICS-5000 with a quaternary pump, a CarboPac PA1 4x250 mm column with a corresponding 4x50 mm guard column and pulsed amperometric detection (Thermo Fisher). The gradient used was: A = 1 mM NaOH in degassed water; C = 1 mM NaOH, 1M NaOAc in degassed water; 0 min ; 20 min ; 60 min ; 63 min ; 83 min ; 87 min ; 95 min ; 97 min ; 105 . Mixtures of commercial or synthetic standards (100-400 μM) were used.5, Ac4ManNAl  or DMSO vehicle. Cells were grown for another 20 h, harvested in a V-shaped 96 well plate and washed twice with 2% FBS in PBS .K-562 cells stably transfected with pSBtet-WT-hAGX1-BH-T1, pSBtet-mut-hAGX1-WT-T1, pSBtet-mut-hAGX1-BH-T1 pSBtet-WT-hAGX1-BH-T2, pSBtet-mut-hAGX1-WT-T2, pSBtet-mut-hAGX1-BH-T2 were seeded into 6-well plates at a density of 500,000 cells/2 mL in growth medium without hygromycin and treated with 0.5 μg/mL doxycycline or left untreated. Cells were treated with doxycycline again after 24 h. After another 24 h, cells were counted, harvested and seeded at a density of 50,000 cells/0.2 mL (flow cytometry) or 200,000 cells/0.6 mL (in-gel fluorescence) fresh growth medium. Cells were treated with doxycycline again, and either caged GalNAc-1-phosphate analog 4, 300 μM BTTAA , 2.5 mM sodium ascorbate, 2.5 mM aminoguanidinium chloride and 50 μM CF680 picolyl azide in cell buffer (35 μL), and incubated for 7 min at room temperature on an orbital shaker. The reaction was quenched by addition of 3 mM bathocuproinedisulfonic acid in PBS (35 μL). Cells were harvested, washed twice with Cell Buffer and once with PBS, and resuspended in ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl2, and 100 mU/μL benzonase (Merck) containing cOmplete protease inhibitors, 0.1 mL). Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . The supernatant was transferred to a new plate and protein concentration was measured by BCA. For enzymatic digest, equal amounts of protein  were diluted to 40 μL with Lysis Buffer (PNGase F digest) or PBS (StcE digest), treated with PNGase F  or StcE (50 nM) and incubated for 12 h at 37 °C , treated with a solution of 50 μM CuSOat 37 °C . The rea4, 300 μM BTTAA, 2.5 mM sodium ascorbate, 2.5 mM aminoguanidinium chloride and 50 μM MB488 picolyl azide (Click Chemistry Tools) in Cell Buffer (50 μL), and incubated for 5 min at room temperature on an orbital shaker. The reaction was quenched by addition of 3 mM bathocuproinedisulfonic acid in PBS (50 μL). Cells were harvested, washed twice with cell buffer and once with PBS, Cells were then fixed with 0.5% (v/v) paraformaldehyde in PBS (100 μL) for 10 min at room temperature in the dark, harvested and washed with cell buffer once. Cells were permeabilized with 0.5% (v/v) Tween-20 in cell buffer (100 μL) for 10 min at room temperature. Cells were harvested, treated with mouse anti-VSV-G-tag (1:200) in 0.5% (v/v) Tween-20 in cell buffer (50 μL) for 30 min at room temperature, and washed with 0.1% (v/v) Tween-20 in cell buffer. Cells were treated with anti-mouse IgG 647  in 0.5% (v/v) Tween-20 in cell buffer (50 μL) for 30 min at room temperature, harvested, washed with 0.1% (v/v) Tween-20 in Cell Buffer and Cell Buffer without detergent. Flow cytometry was performed on an Accuri C6 flow cytometer .For flow cytometry, cells were resuspended in cell buffer (50 μL) and treated with a solution of 50 μM CuSO5 (50 μM) or DMSO vehicle. Cells were grown for another 20 h, harvested and washed with PBS (5 mL). Cell pellets were stored at −80 °C until use.K-562 cells stably transfected with pSBtet-mut-hAGX1-WT-T1, pSBtet-mut-hAGX1-BH-T1, pSBtet-mut-hAGX1-WT-T2, or pSBtet-mut-hAGX1-BH-T2 were seeded into a T75 flask at 5,000,000 cells/15 mL in growth medium without hygromycin, and treated with 0.5 μg/mL doxycycline. Cells were treated with 10 mL growth medium and 0.5 μg/mL doxycycline after 24 h. After another 24 h, cells were counted, harvested and seeded at a density of 10,000,000 cells/25 mL in growth medium without hygromycin. Cells were treated with doxycycline again, and either caged GalNAc-1-phosphate analog Cell pellets were resuspended in 500 μL Lysis Buffer (PBS with 1% (v/v) RapiGest and 1x Calbiochem Protease Inhibitors, Set III) and homogenized using a finger tip sonicator  on ice. Five to six mg protein per sample were treated with PNGase F  dilution in PBS) and incubated for 4 h at 37 °C. The sample was diluted with PBS to a protein content of 2 mg/mL. Beads  were washed with PBS twice (harvest 30 s at 1000 g in table top) in low-bind tubes (Eppendorf) and added to the lysate. Samples were incubated for 16 h at room temperature under rotation. The beads were harvested and the supernatant was discarded. The beads were washed sequentially with 0.1% RapiGest in PBS (3x), 6 M urea in PBS (3x), and PBS (2x), and resuspended in PBS (200 μL). Beads were treated with 10 μL 100 mM DTT (Thermo Fisher) in PBS (10 μL), and incubated at r.t. while shaking (950 rpm) for 30 min. Then, 500 mM iodoacetamide (Sigma Aldrich) in PBS (4 μL) was added and samples were shaken for another 30 min in the dark. Beads were harvested and washed with PBS and 50 mM ammonium bicarbonate in LC/MS-grade water . Beads were resuspended in ABC buffer (200 μL), treated with RapiGest to a final concentration of 0.05% (v/v), and trypsin (1.5 μg in 3 μL ABC buffer). Samples were shaken at 37 °C for 2-3 h, and another 1.5 μg trypsin was added. The reactions were shaken overnight at 37 °C. The beads were harvested, washed with ABC (200 μL) and with LC-MS grade water (3x200 μL) Supernatants and washes were combined and centrifuged . The supernatants were transferred to new tubes and concentrated by Speedvac. Samples were desalted by C18 Zip tips, using 0.1% aq. formic acid (FA) as washing solution and stepwise elution with 50% MeCN/water with 0.1% FA (v/v) and 100% MeCN with 0.1% (v/v) FA.Samples were analyzed by online nanoflow LC-MS/MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher) coupled to a Dionex Ultimate 3000 HPLC (Thermo Fisher). A portion of the sample (6.5 μL out of 8 μL for glycopeptide fractions and 2 μL out of 10 μL for peptide fractions) was loaded via autosampler isocratically onto a C18 nano pre-column using 0.1% formic acid in water (“Solvent A”). For pre-concentration and desalting, the column was washed with 2% ACN and 0.1% formic acid in water (“loading pump solvent”). Subsequently, the C18 nano pre-column was switched in line with the C18 nano separation column (75 μm x 250 mm EASYSpray (Thermo Fisher) containing 2 μm C18 beads) for gradient elution. The column was held at 40 °C using a column heater in the EASY-Spray ionization source (Thermo Fisher). The samples were eluted at a constant flow rate of 0.3 μL/min using a 90 minute gradient and a 140 minute instrument method. The gradient profile was as follows  0:3, 3:3, 93:35, 103:42, 104:95, 109:95, 110:3, 140:3. The instrument method used an MS1 resolution of 60,000 at FWHM 400 m/z, an AGC target of 3e5, and a mass range from 300 to 1,500 m/z. Dynamic exclusion was enabled with a repeat count of 3, repeat duration of 10 s, exclusion duration of 10 s. Only charge states 2-6 were selected for fragmentation. MS2s were generated at top speed for 3 s. HCD was performed on all selected precursor masses with the following parameters: isolation window of 2 m/z, 28%–30% collision energy, orbitrap  detection, and an AGC target of 1e4 ions. ETD was performed if (a) the precursor mass was between 300-1000 m/z and (b) 2 of 10 glyco and/or fingerprint ions  were present at ± 0.1 m/z and greater than 5% relative intensity. ETD parameters were as follows: calibrated charge-dependent ETD times, 2e5 reagent target, precursor AGC target 1e4.Data evaluation was performed with Byonic™ . For protein IDs, Uniprot human proteome  was used as a reference database. Search parameters included semi-specific cleavage specificity at the C-terminal site of R and K, with two missed cleavages allowed. Mass tolerance was set at 10 ppm for MS1s, 0.1 Da for HCD MS2s, and 0.35 Da for ETD MS2s. Methionine oxidation (common 2), asparagine deamidation (common 2), and N-terminal acetylation (rare 1) were set as variable modifications with a total common max of 3, rare max of 1. Cysteine carbamidomethylation was set as a fixed modification. Peptide hits were filtered using a 1% FDR. Additionally, a cut-off value of Log Prob = 5 was set for protein hits.For glycopeptide analysis, search parameters included semi-specific cleavage specificity at the C-terminal site of R and K, with two missed cleavages allowed. Mass tolerance was set at 10 ppm for MS1s, 0.1 Da for HCD MS2s, and 0.35 Da for ETD MS2s. Methionine oxidation (common 2), asparagine deamidation (common 2), and N-terminal acetylation (rare 1) were set as variable modifications with a total common max of 2, rare max of 1. O-glycans were also set as variable modifications (common 2), using a custom database, whereby HexNAc, HexNAc-NeuAc, HexNAc-Hex, HexNAc-Hex-NeuAc, and HexNAc-Hex-NeuAc2 were searched with an additional 287.1371 m/z to account for the chemical modification. HCD was used to confirm that the peptides were glycosylated, whereas ETD spectra were used for site-localization of glycosylation sites. All spectra with these modifications were manually annotated.5 (25 μM) or DMSO vehicle. After another 24 h, the medium was aspirated, and cells were washed with pre-warmed serum-free low-Glc DMEM. Serum-free medium was added, cells were treated with Dox again, and either GalNAc-1-phosphate analog 5 or DMSO vehicle again. Cells were incubated for 20 h. Conditioned supernatant was collected and centrifuged for 5 min at 3000 g. The supernatant was concentrated to 2 mL using an Amicon Ultra-15 centrifuge filter . Samples were stored at −80 °C until further use.HepG2-T1-KO and HepG2-T2-KO cells stably transfected with pSBtet-mut-hAGX1-WT-T1, pSBtet-mut-hAGX1-BH-T1, pSBtet-mut-hAGX1-WT-T2, or pSBtet-mut-hAGX1-BH-T2 were seeded into one 10 cm dish per treatment, using 8 mL growth medium without hygromycin B. Cells were directly induced with 0.5 μg/mL Dox. After 24 h, cells were Dox-induced again and fed with either caged GalNAc-1-phosphate analog 5 and treated the same way.In a repeat experiment, HepG2-T1-KO stably transfected with pSBtet-hAGX13A-T1BH and HepG2-T2-KO cells stably transfected with pSBtet-mut-hAGX1-BH-T2 were treated with GalNAc-1-phosphate analog 4, 2.5 mM sodium ascorbate, 2.5 mM aminoguanidinium chloride, and 50 μM biotin-DADPS-picolyl azide 10.Samples were treated with PNGase F  and incubated for 4 h at 37 °C. Samples were then treated in that order with 600 μM BTTAA, 300 μM CuSOThe click reaction was allowed to proceed for 3 h at r.t. under inversion. The samples were transferred into 15 mL Falcon tubes and treated with 5 mL cold (−20 °C) methanol. Samples were left at −80 °C overnight, when a white precipitate had formed. Samples were centrifuged , the supernatant was discarded, and pellets were washed with 5 mL methanol twice, with centrifugation each time.The supernatant was completely removed and the pellet air-dried. Samples were treated with 250 μL 0.1% (w/v) Rapigest. Samples were sonicated in a water bath for 25 min and centrifuged . The supernatants were transferred to a fresh tube, and pellets were treated with 250 μL 6 M urea in PBS. Samples were sonicated and centrifuged again, the pellets treated with 250 μL PBS, sonicated and centrifuged again. All supernatants from solubilization steps were combined, and samples were diluted with PBS to 2 mL. Protein concentration was determined by BCA.27). Briefly, beads were washed sequentially with 0.1% RapiGest in PBS (2x), 6 M urea in PBS (3x), and PBS (3x), and resuspended in PBS (200 μL). Beads were treated with 100 mM DTT (Thermo Fisher) in PBS (10 μL), and incubated at r.t. while shaking (950 rpm) for 30 min. Then, 500 mM iodoacetamide (Sigma Aldrich) in PBS (4 μL) was added and samples were shaken for another 30 min in the dark. Beads were harvested and washed with PBS and 50 mM ammonium bicarbonate in LC/MS-grade water . Beads were resuspended in ABC buffer (200 μL), treated with RapiGest to a final concentration of 0.05% (w/v), and trypsin (0.5 μg in 1 μL ABC buffer). Samples were shaken at 37 °C for 2-3 h, and another 0.5 μg trypsin was added. The reactions were shaken overnight at 37 °C. The beads were harvested, washed with ABC (200 μL) and with LC-MS grade water (3x200 μL). Supernatants were discarded, and the beads were treated with 2% (v/v) formic acid in water (150 μL). Samples were shaken at r.t. for 30 min, and the cleavage step was repeated. The beads were washed with water (100 μL) and 1:1 MeCN/2% aq. FA (100 μL). All supernatants were combined for each sample, centrifuged  and concentrated by Speedvac. Samples were dissolved in 0.1% (v/v) formic acid in water and used for mass spectrometry (see above).Beads  were washed with PBS twice (harvest 30 s at 1000 g in table top) in low-bind tubes (Eppendorf) and treated with the protein solutions. Samples were incubated for 16 h at room temperature under rotation. The beads were harvested and the supernatant was discarded. The beads were treated as described . Spots were developed with sugar stain (0.1% (v/v) 3-methoxyphenol, 2.5% (v/v) sulfuric acid in EtOH) or ceric ammonium molybdate stain (5% (w/v) ammonium molybdate, 1% (w/v) cerium(II) sulfate and 10% (v/v) sulfuric acid in water) dipping solutions. Flash chromatography was carried out on Fluka Kieselgel 60 (230-400 mesh). Solvents were removed under reduced pressure using a rotary evaporator and high vacuum (1 mbar).1H, 13C and 2D NMR spectra were measured with a Varian AS400 spectrometer or a Varian AS600 spectrometer at 25°C. Chemical shifts (σ) are reported in parts per million (ppm) relative to the respective residual solvent peaks . Two-dimensional NMR experiments  were performed to assign peaks in 1H spectra. The following abbreviations are used to indicate peak multiplicities: s singlet; d doublet; dd doublet of doublets; dt doublet of triplets; m multiplet. Coupling constants (J) are reported in Hertz (Hz). High resolution mass spectrometry by electrospray ionization (ESI-HRMS) was performed at Stanford University Mass Spectrometry, with a micrOTOF-Q II hybrid quadrupole time-of-flight mass spectrometer (Bruker) equipped with an Agilent 1260 UPLC.SI-1  in MeCN (0.5 mL) were added at 0°C phosphoramidite SI-2  solution in MeCN). The reaction was warmed to room temperature and stirred for 1 h. Phosphoamidite SI-2  and 1H-tetrazole  were added to drive the reaction to completion. The mixture was stirred for 20 min at room temperature, cooled to 0 °C and treated with mCPBA . The reaction was stirred for 2 h at that temperature and quenched with 10% aq. Na2SO3 (10 mL). The solution was extracted with CH2Cl2 (5x10 mL), the combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by flash chromatography (hexanes/EtOAc 1:0 to 1:1 with 0.5% (v/v) NEt3) to give phosphotriester 5  as a clear oil. Rf (hexanes/EtOAc with 0.5% (v/v) NEt3, TLC plates pre-neutralized with hexanes/2% NEt3) = 0.25.To a stirred solution of lactol ite SI-2   in MeCN (0.73 mL) were added at 0 °C phosphoramidite SI-2  in 0.4 mL toluene and 1H-tetrazole  solution in MeCN). The reaction was warmed to room temperature and stirred for 1 h. Phosphoamidite SI-2  was added to drive the reaction to completion. The mixture was stirred for 20 min at room temperature, cooled to −20 °C and treated with mCPBA . The reaction was stirred for 2 h at that temperature and quenched with 10% aq. Na2SO3 (10 mL). The solution was extracted with CH2Cl2 (5x10 mL), the combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by flash chromatography (hexanes/EtOAc 1:0 to 5:1 to 3:1to 1:1 with 0.5% (v/v) NEt3) and size exclusion chromatography  to give phosphotriester 6  as a clear oil.To a stirred solution of lactol SI-4  in MeCN (0.73 mL) were added at 0 °C phosphoramidite SI-2  in 0.4 mL toluene and 1H-tetrazole  solution in MeCN). The reaction was warmed to room temperature and stirred for 1 h. Phosphoamidite SI-2  was added to drive the reaction to completion. The mixture was stirred for 20 min at room temperature, cooled to −20 °C and treated with mCPBA . The reaction was stirred for 2 h at that temperature and quenched with 10% aq. Na2SO3 (10 mL). The solution was extracted with CH2Cl2 (5x10 mL), the combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by flash chromatography (hexanes/EtOAc 1:0 to 5:1 to 3:1to 1:1 with 0.5% (v/v) NEt3) and size exclusion chromatography  to give phosphotriester 7  as a clear oil.To a stirred solution of lactol SI-5  in 5:2 MeOH/water (1.5 mL) was added at room temperature triethylamine (0.3 mL). The reaction was warmed to 50 °C and stirred for 20 h at that temperature, when the starting material was converted to a lower-running spot (TLC CH2Cl2/MeOH 10:1). The solvents were evaporated, and the residue was co-evaporated with toluene (3x5 mL) to give the intermediate triethylammonium salt as a yellow oil.To a stirred solution of ester 2Cl2 (1.5 mL) were added at room temperature triethylamine , EDC hydrochloride  and N-hydroxysuccinimide . The mixture was stirred for 2 h, and 3-amino-1-propanol SI-6  was added. The reaction was stirred for 20 h, diluted with CH2Cl2 (5 mL), and filtered through cotton wool. The solution was concentrated, and the residue was purified by flash chromatography (CH2Cl2/MeOH 99:1 to 95:5) to give amide SI-7  as a clear oil.To a stirred solution of the intermediate triethylammonium salt in CHSI-10  in 5:2 CH2Cl2 (2.6 mL) were added at room temperature active ester SI-9  and triethylamine . The reaction was stirred for 16 h and treated with EDC hydrochloride . The mixture was stirred for 48 h, the reaction was diluted with EtOAc (10 mL), washed with 1 N HCl (2x5 mL), sat. aq. NaHCO3 (5 mL) and brine (5 mL). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by flash chromatography  as a clear oil.To a stirred solution of ammonium salt SI-8  in CH2Cl2 (0.5 mL) were added at room temperature triethylamine  and dichlorodiphenylsilane . The mixture was stirred for 3 h, and alcohol SI-7  was added. The reaction was stirred for 20 h, quenched with sat. aq. NaHCO3 (3 mL) and diluted with CH2Cl2 (5 mL). The layers were separated, and the aqueous phase was extracted with CH2Cl2 (5x5 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography  and size exclusion chromatography  to give diphenyldisiloxane 10  as a clear oil.To a stirred solution of alcohol P values are indicated in figures.The number of replicates for each experiment is specified in Figure legends. Flow cytometry experiments were run in three independent replicates on three different days. Statistical analysis was performed by two-tailed ratio paired t test, and


N,N′-dioxide metal complexes, delivering a broad range of optically pure 1,5-dicarbonyl compounds with two vicinal tertiary carbon stereocenters in up to >99% ee under mild conditions. Meanwhile, stereodivergent synthesis is disclosed to yield all four stereoisomers of products. Control experiments suggest an isomerization process involved in the reaction and give an insight into the role of NEt3. In addition, Mannich reaction and sulfur-Michael addition of β,γ-unsaturated 2-acyl imidazoles proceed smoothly as well under the same catalytic system.The investigation of diverse reactivity of β,γ-unsaturated carbonyl compounds is of great value in asymmetric catalytic synthesis. Numerous enantioselective transformations have been well developed with β,γ-unsaturated carbonyl compounds as nucleophiles, however, few example were realized by utilizing them as not only nucleophiles but also electrophiles under a same catalytic system. Here we report a regioselective catalytic asymmetric tandem isomerization/α-Michael addition of β,γ-unsaturated 2-acyl imidazoles in the presence of chiral  The investigation of reactivity of β,γ-unsaturated carbonyl compounds is of great synthetic value, especially in asymmetric transformations. Here, the authors report a catalytic asymmetric tandem isomerization/α-Michael addition of β,γ-unsaturated 2-acyl imidazoles in presence of chiral N,N′-dioxide metal catalysts. Especially, γ-addition as dienolate pronucleophiles with either metal catalysis28 or organocatalysis36 has been widely documented during the past several years, and the maintained π-conjugation of γ-addition process leading to thermodynamically stable conjugated products  bidentate coordination with a Lewis acid of acyl imidazole exhibits good stereocontrol55 and (2) the strong coordination facilitates isomerization of the β,γ-unsaturated ketone to an α,β-unsaturated ketone. Chiral N,N′-dioxide-metal59 complexes catalyze diverse reactions of β,γ-unsaturated 2-acyl imidazoles, including tandem isomerization/α-Michael addition 2, and Mg(OTf)2; however, only trace amount of the self-α/β-addition product 2a was observed, which was generated from α-addition of E-1a with the corresponding α,β-unsaturated 2-acyl imidazole upon C=C isomerization . Lanthanide metal salts La(OTf)3 and Yb(OTf)3 could also mediate the reaction but gave lower yields and ee values (entries 3 and 4). The screening of chiral backbones and steric hindrance of the amide moiety on the N,Nʹ-dioxide ligands afforded no better results . To our delight, the diastereoselectivity could be improved to 10:1 with addition of NEt3 (entry 6). Other common chiral ligands such as Box, Pybox, and BINAP were also explored, and 32% yield, 5:1 dr with 60% ee were observed as the best results  were converted into the corresponding dimerization products 2a–2j in good yields (65–81%), high diastereoselectivities (7.5:1 to 11:1), and excellent ee values (97–>99%). Furthermore, β,γ-unsaturated carbonyl compounds containing 3-thienyl, N-methyl-5-indolyl and 2-naphthyl moieties were also proven to be suitable substrates, affording 2k–2m with good results . Moreover, aliphatic-substituted β,γ-unsaturated 2-acyl imidazoles exhibited high tolerance as well, generating the desired products 2n–2q with a high level of yields (63–84%) and stereoselectivities . Estrone-derived 1r could be transformed into 2r smoothly in 69% yield, 2.8:1 E/Z, >19:1 dr, and 99% ee for E-isomer. Other Michael acceptors such as α,β-unsaturated 2-acyl imidazole and ethyl vinyl ketone were aslo suitable in this reaction, delivering 2s–2w with good yields (60–71%) and stereoselectivities . The absolute configuration of 2j was determined to be  by X-ray crystallography analysis.The generality of the tandem isomerization/α-Michael addition reaction was investigated under the optimized conditions Fig. . An arra3 were explored as the electrophiles. By switching the catalyst to La(OTf)3/L3-PitBu complex  except for 4p with 52% ee  and high enantioselectivities (85–98% ee) as single isomers  by X-ray crystallography analysis.The reaction described above indicated that β,γ-unsaturated 2-acyl imidazoles performed both α-addition reaction and β-addition upon isomerization under proper Lewis acid catalysts. Next, to extend the scope of α-addition of β,γ-unsaturated 2-acyl imidazoles, several types of imines  ee Fig. . Aldiminers Fig. . The abs6a was achieved if E-1a reacted with thiophenol 5a. After examination of the reaction conditions  in 39–95% yields with 70–93% ee values. For the Michael acceptors, aryl- and alkyl-substituted β,γ-unsaturated 2-acyl imidazoles were also tolerated in this reaction, giving 6j–6p in 60–92% yields with 80–92% ee.Inspired by the isomerization process of β,γ-unsaturated 2-acyl imidazoles into α,β-unsaturated 2-acyl imidazoles, we next enlarged the diverse reactivity of β,γ-unsaturated compounds as the electrophiles under the current catalytic system. However, only a trace amount of desired tandem isomerization/sulfur-Michael addition product 2a was conducted. The current reaction could be carried out at 7.0 mmol scale without loss of yield (70%), diastereoselectivity (10:1 dr), and ee value (98%)  Fig. . Further ee Fig. . Chiral  ee Fig. 69.Fig. 63 led to an increase in diastereoselectivity  under the standard conditions for 12 h , excellent diastereoselectivities (19:1 to >19:1), and 98% ee within 2 h no matter with or without addition of NEt3  and diastereoselectivities (8:1–>19:1) with excellent ee values by matching the E/Z-configurated 10 and the chiral ligand . Then, the β,γ-unsaturated ketone E-1a attaches to Y* as a dienolate in the presence of NEt3 to give the intermediate T1, and which partly transforms into the α,β-unsaturated ketone E/Z-10 upon 1,5-proton shift. Next, the catalyst-bonded dienolate will react with the newly formed Michael acceptors. The α-Re-face of β,γ-unsaturated 2-acyl imidazole E-1a is strongly shielded by the nearby aryl ring of the ligand. Therefore, the dienolate prefers to attack E/Z-10 from its α-Si-face (T2). Finally, the desired product 2a dissociates after a protonation of the intermediate T3, and the catalyst is regenerated to accomplish one catalytic cycle.Based on the absolute configuration of the product sed Fig. . First, In summary, we have disclosed the diverse transformation of β,γ-unsaturated 2-acyl imidazoles in the presence of chiral Lewis acid catalysts, involving catalytic asymmetric tandem isomerization/α-Michael addition, sulfur-Michael addition, and direct Mannich reaction. A wide range of chiral 1,5-dicarbonyl and functionalized carbonyl compounds was afforded with good to excellent levels yields, diastereoselectivities, and enantioselectivities. The β,γ-unsaturated 2-acyl imidazoles features various reactivities, acting as both α-nucleophile and β-electrophile upon isomerization, which provides a route for conjugate addition of unstable α,β-unsaturated carbonyl compounds. Meanwhile, all four stereoisomers with two vicinal tertiary stereocenters could be prepared by matching the configuration between substrates and chiral ligand. Besides, the desired products could be easily transformed into useful compounds with good results under mild conditions. Further studies on this methodology are ongoing.3 (0.005 mmol), L3-RaPr2 (0.005 mmol), β,γ-unsaturated 2-acyl imidazole E-1a (0.20 mmol), and NEt3 (0.02 mmol) were dissolved in 1.0 mL of toluene under N2 atmosphere. The mixture was stirred at 25 °C for 12 h and subjected to column chromatography on silica to afford the product 2a (Pet/EtOAc = 1:1 as eluent) as a colorless foam.Y(OTf)L3-PitBu , La(OTf)3 , 3 Å M.S. (30 mg), and E-1a  in CH2ClCHCl2 (1.0 mL). The mixture was stirred at 30 °C for 30 min, and then 3a  was added at 0 °C. After 3a was consumed (detected by thin-layer chromatography (TLC)), the residue was purified by column chromatography on silica gel to afford the product 4a (Pet/EtOAc = 1:1 as eluent) as a colorless foam.A dry reaction tube was charged with L3-RaPr2 , La(OTf)3 , E-1a , and pyrazolinone-derived ketimine  in CHCl3 (1.0 mL). After ketimine was consumed (detected by TLC), the residue was purified by column chromatography on silica gel to afford the product 4i (Pet/EtOAc =  2:1 as eluent) as a colorless foam.A dry reaction tube was charged with L3-RaPr2 , La(OTf)3 , E-1a , 4 Å M.S. (20 mg), and benzaldehyde-dervived aldimine  in CH2ClCHCl2 (1.0 mL). After E-1a was consumed (detected by TLC), the residue was purified by column chromatography on silica gel to afford the product 4r (Pet/EtOAc = 2:1 as eluent) as a colorless oil.A dry reaction tube was charged with L3-PePr3 , Dy(OTf)3 , and Z-1a  in CH2ClCHCl2 (1.0 mL). PhSH (0.10 mmol) was added and the mixture was stirred at 25 °C for 17 h. After PhSH was consumed (detected by TLC), the residue was purified by column chromatography on silica gel to afford the product 6a (Pet/EtOAc = 3:1 as eluent) as a pale yellow oil.A dry reaction tube was charged with Supplementary InformationPeer Review File


The title molecules were synthesized by the reaction of the corresponding sodium benzoate with chloroacetic acid amide. Single crystals were obtained from the reaction products under the same conditions. 9H8BrNO3, I, 2-amino-2-oxoethyl 4-nitrobenzoate, C9H8N2O5, II, and 2-amino-2-oxoethyl 4-aminobenzoate monohydrate, C9H10N2O3·H2O, III, are 86, 78 and 88%, respectively. The low yield of II is explained by the reduced reactivity of the molecule in a nucleophilic exchange reaction because of the negative induction and negative mesomeric effects of the nitro group on the benzene ring. Single crystals were obtained from the products under the same (temperature and solvent) conditions. In the case of III, the crystals formed as a monohydrate. In all three crystal structures, the same type of intermolecular N—H⋯O hydrogen bonds are observed, but the molecules differ in some torsion angles as well as in the dihedral angles between the mean planes of the benzene rings and the amide groups.The title molecules were synthesized by the reaction of 4-substituted sodium benzoates with chloroacetic acid amide in the presence of dimethylformamide. The yields of 2-amino-2-oxoethyl 4-bromobenzoate, C Molecules containing an aromatic ring, a carboxyl and an amino group represent an important class of organic compounds and, with several reaction centers, they are important intermediates in industry. They are often used as synthons in organic synthesis and are also widely used as ligands in the coordination chemistry of various transition metals. These ligands can form a variety of complex compounds as they possess several Lewis base sites.I), 2-amino-2-oxoethyl 4-nitrobenzoate (II) and 2-amino-2-oxoethyl)-4-aminobenzoate monohydrate (III)  Fig. 1 were synI consists of two independent molecules (A and B), which differ in the position of the amide groups relative to the benzoate (r.m.s. deviations of 0.021 Å for A and 0.031 Å for B) fragments, as indicated by the dihedral angles of 82.5 (4) and 75.9 (3)° in A and B, respectively. The asymmetric unit of II contains only one molecule of 2-amino-2-oxoethyl 4-nitrobenzoate. The dihedral angle between the mean planes of the amide and the benzoate (r.m.s. deviation = 0.070 Å) groups is 89.4 (2)°. The asymmetric unit of III contains one water molecule and one 2-amino-2-oxoethyl 4-aminobenzoate molecule  and amide (O3/C9/N1) units but the dihedral angle between these planes is different in each case because of the torsion angle about the bridging methylene group , B⋯B (N1B—H2B⋯O3Bii) and B⋯A (N1B—H2B⋯O3Biii) interactions cross-link the molecules, generating rings with an et al., 1999A and B molecules contain a bromine atom, a short intermolecular Br⋯Br interaction only occurs between B molecules [Br1B⋯Br1B°. A characteristic intermolecular interaction for II is the formation of centrosymmetric dimers as a result of the N1—H1⋯O3i hydrogen bonds formed between amide fragments  interactions [at 2.874 (4) Å these are 0.14 Å less than the sum of the van der Waals radii] are observed between the nitro groups of the dimers  scaffold in the Cambridge Structural Database  of the sodium salt of p-bromobenzoic acid were added 6 mL of DMF. The resulting mixture was heated for 10 min  and 0.1 g (1 mmol) of chloroacetamide was added. The flask was equipped with a reflux condenser and mechanical stirrer and was heated in a sand bath with stirring at 426 K for 6 h. The DMF was removed in vacuo (15 mm Hg) at 328 K. After cooling, cold water was poured into the reaction mixture to completely eliminate the residual reactants and DMF. The resulting precipitate was filtered off to give 0.22 g (86% yield) of product. RF = 0.65 [in 5:1.5:1 (v/v) CHCl3/C6H6/CH3OH solvent system]; m.p. 475–477 K. 1H NMR : 7.95 , 7.61 , 4.72 .(2-Amino-2-oxoethyl)-4-nitrobenzoate. The reaction yield is 78%. RF = 0.62 [in 5:1.5:1 (v/v) CHCl3/C6H6/CH3OH solvent system]; m.p. 439–441 K. 1H NMR : 8.23 , 8.19 , 4.73 .(2-Amino-2-oxoethyl)-4-aminobenzoate. The reaction yield is 88%. RF = 0.53 [in 5:1.5:1 (v/v) CHCl3/C6H6/CH3OH solvent system]; m.p. 435–438 K. 1H NMR : 7.75 , 6.58 , 4.62 .Each compound was dissolved in ethanol and the solvent allowed to evaporate at room temperature. Colourless crystals suitable for X-ray diffraction analysis were obtained.The crystal of the 2-amino-2-oxoethyl 4-aminobenzoate monohydrate loses its transparency without chemical change (without becoming amorphous) in the range 344–346 K when the crystals are heated and melts in the range 435–438 K.9H8BrNO3, I, 2-amino-2-oxoethyl 4-nitrobenzoate, C9H8N2O5, II, and 2-amino-2-oxoethyl 4-aminobenzoate monohydrate, C9H10N2O3·H2O, III, are 86, 78 and 88%, respectively. The low yield of II is explained by the reduced reactivity of the molecule in a nucleophilic exchange reaction because of the negative induction and negative mesomeric effects of the nitro group on the benzene ring.The yields of 2-amino-2-oxoethyl 4-bromobenzoate, C2 and 0.93 Å for Car) and included in the refinement as riding contributions with Uiso(H) = 1.2Ueq(C) [Uiso = 1.5 Ueq(C) for methyl H atoms]. The hydrogen atoms attached to N and O (water) were located in difference-Fourier maps and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989020014371/mw2171sup1.cifCrystal structure: contains datablock(s) I, II, III, Global. DOI: 10.1107/S2056989020014371/mw2171Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020014371/mw2171IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020014371/mw2171IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989020014371/mw2171Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014371/mw2171IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014371/mw2171IIIsup7.cmlSupporting information file. DOI: 2041177, 2041176, 2041175CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Secondary hyperparathyroidism (SHPT) after obesity surgery may affect bone health. Optimal vitamin D levels have not been established to prevent SHPT postoperatively. We investigated whether SHPT differed across threshold levels of serum 25-hydroxyvitamin D (S-25(OH)D) from 6 months up to 5 years after Roux-en-Y gastric bypass (RYGB).We included 554 patients at follow-up 5 years postoperatively. Blood samples were analysed for S-25(OH)D, ionized calcium (iCa) and parathyroid hormone (PTH) during follow-up.P < 0.001). PTH and SHPT development are related with S-25(OH)D, and PTH differed between all subgroups of S-25(OH)D. SHPT occurred less frequently across all subgroups of S-25(OH)D ≥ 50 nmol/l during follow-up: odds ratio (OR) 0.44 (95% CI 0.36–0.54) in patients with S-25(OH)D ≥ 50 nmol/l, OR 0.38 (0.30–0.49) with S-25(OH)D ≥ 75 nmol/l and OR 0.19 (0.12–0.31) with S-25(OH) D ≥ 100 nmol/l, all compared with S-25(OH)D < 50 nmol/l. At 5 years, 208/554 patients (38%) had SHPT; SHPT was found in 94/188 patients (50%) with S-25(OH)D < 50 nmol/l, in 69/222 (31%) with S-25(OH)D 50–74 nmol/l, in 40/117 (34%) with S-25(OH)D 75–99 nmol/l and in 5/27 (19%) with S-25(OH)D ≥ 100 nmol/l. An interaction existed between S-25(OH)D and iCa. Bone alkaline phosphatase remained increased with SHPT.PTH and prevalence of SHPT increased from 6 months to 5 years postoperatively, while S-25(OH)D and iCa decreased D and development of PTH and SHPT. The prevalence of SHPT was lower with threshold levels 25(OH)D ≥ 50 nmol/l and ≥ 75 nmol/l over the 5 years, and lowest with S-25(OH)D ≥ 100 nmol/l.The online version of this article (10.1007/s11695-020-04582-5) contains supplementary material, which is available to authorized users. Obesity surgery provides effective weight loss in morbid obesity , 2. ChanRelationships of calcium and vitamin D with PTH should be explored further, as PTH may impact bone turnover and bone mineral density (BMD) postoperatively –24. MoreWe aimed to study the development of PTH and SHPT over 5 years after Roux-en-Y gastric bypass (RYGB), and relationships between these and different threshold levels of S-25(OH)D ≥ 50 nmol/l. We assessed whether the prevalence of SHPT would be lower among patients with higher S-25(OH)D up to 5 years postoperatively.This longitudinal observational cohort study was analysed prospectively, and the report was written to comply with the STROBE checklist .2, or BMI ≥ 35 kg/m2 with obesity-related comorbidities [Morbid obesity was defined as BMI ≥ 40 kg/mbidities . Obesitybidities , 30.We aimed for minimum 500 patients in a population with high follow-up. Candidates were RYGB patients operated 2004–2009. They were evaluated preoperatively and postoperatively with weight, height and blood samples. Follow-up visits were after 6 weeks, 6 months, 1 year, 2 years, 3–4 years and 5 years. At 5 years, all were contacted by letter and eventually by telephone. Body weight was measured electronically , height with wall-fixed steel measure and blood samples were drawn after overnight fast.Patients with signed consent and valid PTH and S-25(OH)D at 5 years were candidates, while patients with primary hyperparathyroidism and elevated creatinine were excluded.2+/pH Analyser (Bayer); from December 2005 to December 2007, (b) Rapidlab 348 pH/Blood Gas Analyser (Instru-Med) ; from January 2008 to January 2012, (c) Rapidlab 348 pH/Blood Gas Analyser (Instru-Med) (reference range 1.18–1.35 mmol/l); from February 2012, (d) Cobas b221 (Roche Diagnostics) . Reference for iCa was (c) with reference range 1.18–1.35 mmol/l. Values from (a) and (b) were transformed to (c) adding 0.015 mmol/l (differences between reference ranges). Similarly were values from (d) transformed to (c) adding 0.025 mmol/l. B-ALP was determined enzymatically after immune-extraction (Metra Biosystems) . Standard analyses included S-25(OH)D, iCa and PTH during follow-up, which were analysed sporadically preoperatively. Phosphate, magnesium, creatinine and total alkaline phosphatase (ALP) in plasma were analysed at the Central Laboratory, Oslo University Hospital, Aker, with a Modular (Roche) analyzer: Phosphate and magnesium were determined photometrically, creatinine enzymatically and ALP by an enzymatic calorimetric measurement.Serum intact PTH (1–84), S-25(OH)D, ionized calcium (iCa) and bone specific alkaline phosphatase (B-ALP) were determined at the Hormone Laboratory, Oslo University Hospital, Aker. PTH was analysed by a chemiluminoimmunometric assay   7%). S-25(OH)D was determined by radioimmunoassay (Dia-Sorin) , from September 2012 with liquid chromatography-mass spectrometry (LC-MS/MS) ; measurements were comparable. Different methods were used for iCa: first, (a) CIBA Corning, instrument 634 CaSHPT was defined as PTH > 7.0 pmol/l with no elevation of iCa (i.e. ≤ 1.35 mmol/l). S-25(OH)D was grouped into 4 categories, < 50, 50–74, 75–99 and ≥ 100 nmol/l. S-25(OH)D < 50 nmol/l is commonly acknowledged as vitamin D deficiency (or insufficiency) . CalciumRecommended daily supplements included one multivitamin  and two combination tablets, each containing calcium carbonate 500 mg and cholecalciferol 400 IU. Compliance was defined by use of calcium ≥ 500 mg and vitamin D ≥ 600 IU minimum 5 days a week, noncompliance as less or no use. Supplements were adjusted to keep blood values within normal reference range, from 2012 to maintain S-25(OH)D ≥ 50 mmol/l, or S-25(OH)D ≥ 75 nmol/l in cases with SHPT . Our supt test and chi-square test as appropriate. Regression analyses were performed with linear mixed model, diagonal covariance matrices for PTH and B-ALP using individual repeated measurements, time-dependent covariates and random intercept. Variables were included in multivariate analyses of PTH with stepwise backward elimination of nonsignificant variables. Gender, age and BMI were included as covariates. We tested multiplicative interactions for S-25(OH)D, iCa and time on PTH, and PTH and time on B-ALP. B-ALP was adjusted for gender. Generalized estimating equations (GEE), unstructured covariance matrices were used for SHPT. Missing data were not imputed. We analysed two periods: from baseline to 6 months postoperatively and from 6 months to 5 years. The second period was the main focus, as S-25(OH)D and PTH were routinely assessed. Continuous variables are presented with means and standard deviations (± SD), categorical variables in percentages, and odds ratios (OR) and relative ratios (RR) with 95% confidence intervals (95% CI).Statistical analyses were performed with IBM SPSS for Windows, version 25. Continuous and categorical variables were tested with Of 823 operated, 584 71%) attended 5-year follow-up. Included were 554 patients (67%), after exclusion of 4 with no signed consent, 10 with a suspicious primary hyperparathyroidism, 3 with elevated creatinine, and 13 with missing data of S-25(OH)D and PTH at 5 years. Follow-up period was 5.3 ± 0.4 years. Three patients had moved from our region and could not be contacted, and 7 had died during the 5 years. Table % attende2, PTH decreased from 6.4 ± 2.8 to 5.2 ± 2.5 pmol/l and prevalence of SHPT from 35 to 18% . S-25(OH)D and iCa were independently related with PTH development (P < 0.001).BMI decreased 27% in the first 6 months from 46.2 ± 5.3 to 33.8 ± 5.1 kg/mP < 0.001). PTH related positively with B-ALP (P < 0.001).PTH decreased in all subgroups of S-25(OH)D. Patients with S-25(OH)D < 50 nmol/l had highest PTH preoperatively and 6 months postoperatively, and they had the largest decrease in PTH (P < 0.001). BMI increased to 34.5 ± 6.2 kg/m2 (P = 0.005).Figure S-25(OH)D was inversely related with development of PTH and occurrence of SHPT from 6 months to 5 years P < 0.001), and PTH differed between all subgroups of S-25(OH)D (P < 0.001) D , but SHPT not significantly (RR 0.57 (0.25–1.28)) at 5 years. These relationships were not significant with iCa above lower range (iCa ≥1.24 mmol/l).At 5 years, 208 of 554 patients 38%) had SHPT. The prevalence was 50% with S-25(OH)D < 50 nmol/l   increased from 26% to 61% (01) Fig. . PTH incAn interaction existed between S-25(OH)D and iCa. At 5 years, SHPT was most prevalent with combined vitamin D deficiency and iCa in the lower range, and there was no SHPT with S-25(OH)D ≥ 100 nmol/l and iCa above the lower range Fig. .In multivariate analyses of PTH from 6 months to 5 years Table , the relP < 0.001), but not significantly at 5 years.360 of 475 (77%) used supplements of calcium and vitamin D at 6 months, and 278 (50%) at 5 years. S-25(OH)D was higher and PTH lower in users over the period (P = 0.004).PTH related positively with B-ALP and ALP over the 5 years (P < 0.001), with interaction for PTH and time. B-ALP declined by 1.3 U/l per year, but remained increased with SHPT (P < 0.001 for group difference). Total ALP declined slower with SHPT D was related with PTH development. Patients with vitamin D deficiency had the largest increase in PTH from 6 months, while patients with high S-25(OH)D ≥ 100 nmol/l had lowest PTH and prevalence of SHPT.This study is among few longitudinal long-term reports of SHPT by vitamin D and calcium status after RYGB. Vitamin D deficiency is usually defined by S-25(OH)D < 30–50 nmol/l, while target levels of S-25(OH)D ≥ 50–75 nmol/l are adopted in most recommendations after obesity surgery , 2, 4–8.The observed increase in PTH from 6 months is in accordance with other long-term evaluations –13. S-25Calcium absorption seems reduced after RYGB , 32. HowOur observations suggest a role of iCa on PTH. The proportion of patients with iCa in the lower range increased over time, and iCa was related with PTH development. This relationship was independent of S-25(OH)D. An interaction existed between S-25(OH)D and iCa on PTH. Still, iCa declined in all subgroups of S-25(OH)D but more slowly with higher levels.Several studies have failed to document benefits of vitamin D and calcium supplementation, and the regimens have been questioned , 24. ThiWe also found higher B-ALP in patients with SHPT up to 5 years postoperatively, suggesting higher bone turnover. SHPT might therefore help explain increased bone turnover, which is observed up to 5 years after RYGB , 19, 22.In clinical practice, SHPT may be considered as a marker of vitamin D and calcium insufficiency, and it is of concern after RYGB. Further research should address whether increasing S-25(OH)D levels can suppress SHPT and improve bone health. Higher doses seem necessary to achieve sufficient vitamin D levels and suppress SHPT after RYGB , 24, 35.Attention to calcium status seems relevant to identify risk for SHPT. SHPT was more frequent with iCa in the lower range. Higher S-25(OH)D can increase calcium levels and lower PTH, and some individuals may need higher vitamin D levels than others , 26, 36.Optimal S-25(OH)D levels are not established after RYGB and obesity surgery in general. Achieving S-25(OH)D ≥ 100 nmol/l may be needed to suppress SHPT more effectively in some individuals, with an aim to improve long-term bone health.The main strength of this study was a large sample size with high 5-year follow-up rate and repeated measurements, providing statistical strength. Bias of primary hyperparathyroidism was minimized. The single centre study design with standard surgical and follow-up procedures strengthen internal validity, but the findings need testing in other populations. As relationships up to 5 years were the primary focus, only patients with 5-year data on S-25(OH)D and PTH were included. Prevalence of SHPT in nonattenders may be higher than observed, assuming less compliance . Data onS-25(OH)D levels related inversely with PTH development and occurrence of SHPT up to 5 years after RYGB. The prevalence of SHPT was lower with S-25(OH)D thresholds ≥ 50 nmol/l and ≥ 75 nmol/l. Some patients may need S-25(OH)D ≥ 100 nmol/l to suppress SHPT more effectively.ESM 1(DOCX 22 kb)


The crystal structures of the fumarate salts of three prodrugs of synthetic psychedelics  are reported. N-ethyl-N-methyltryptammonium (4-AcO-MET) hydrofumarate {systematic name: [2-(4-acetyloxy-1H-indol-3-yl)ethyl](methyl)ethylazanium 3-carboxyprop-2-enoate}, C15H21N2O2+·C4H3O4−, 4-acetoxy-N-allyl-N-methyltryptammonium (4-AcO-MALT) hydrofumarate {systematic name: [2-(4-acetyloxy-1H-indol-3-yl)ethyl](methyl)prop-2-enylazanium 3-carboxyprop-2-enoate}, C16H21N2O2+·C4H3O4−, and 4-acetoxy-N,N-diallyltryptammonium (4-AcO-DALT) fumarate–fumaric acid (1/1) (systematic name: bis{[2-(4-acetyloxy-1H-indol-3-yl)ethyl]diprop-2-enylazanium} but-2-enedioate–(E)-butenedioic acid (1/1)), 2C18H23N2O2+·C4H2O42−·C4H4O4, are reported. All three salts possess a protonated tryptammonium cation. The 4-AcO-MET and 4-AcO-MALT compounds are charge-balanced by 3-carboxyacrylate (hydrofumarate) anions. The 4-AcO-DALT complex crystallizes as a two-to-one tryptammonium-to-fumarate salt, which co-crystallizes with a fumaric acid molecule. Each structure is consolidated by N—H⋯O and O—H⋯O hydrogen bonds.The solid-state structures of the salts of three psilacetin derivatives, namely, 4-acetoxy- N,N,N-trimethyl-4-phosphoryloxytryptamine (C12H17N2O4P), which has recently been cleared for a number of clinical trials after receiving the ‘breakthrough therapy’ designation from the US Food and Drug Administration , or psilocin, which is the active metabolite. Psilocin is a potent serotonin 2a agonist, and is the primary origin of its psychoactive properties , is a synthetic alternative to psilocybin. It also acts as a prodrug of psilocin, with the acetyl group of psilacetin being hydrolysed as it is metabolized, converting 4-AcO-DMT to 4-HO-DMT. Psilacetin is easier to synthesize than psilocybin, and can also be produced at a lower cost, making it, perhaps, a better candidate for the delivery of psilocin , 4-acetoxy-N-allyl-N-methyltryptamine, C16H20N2O2 (4-AcO-MALT), and 4-acetoxy-N,N-diallyltryptamine, C18H22N2O2 (4-AcO-DALT). These variations of psilacetin have garnered very little attention in the scientific literature, with only one reference made to 4-AcO-MET in a chromatographic screening article prior to this year  cation and one hydrofumarate (C4H3O4−) anion. The indole ring system of the cation is near planar with an r.m.s. deviation of 0.015 Å. The hydrofumarate anion is slightly twisted, demonstrating a deviation from planarity of 0.158 Å, and a C16/O3/O4 carboxylate to C19/O5/O6 carboxylic acid plane normal angle of 23.0 (3)°. The N-methyl-N-ethyl group of the cation is disordered over two orientations in a 0.76 (1):0.24 (7) ratio. The ethylammonium arm is turned slightly away from the plane of the indole, with C10—C9—C11—C12 and C10—C9—C11—C12A torsion angles of 39.7 (7) and 49.5 (2)°, respectively, for the two orientations.The molecular structure of 4-AcO-MET hydrofumarate, (I)N-allyl-N-methyltryptammonium (C16H21N2O2+) cation and one hydrofumarate (C4H3O4−) anion. The indole ring system of the compound is almost planar with an r.m.s. deviation from planarity of 0.006 Å. The ethylammonium arm is turned slightly away from the plane of the indole ring, with a C10—C9—C11—C12 torsion angle of 39.8 (4)°. The hydrofumarate anion is slightly twisted, showing a deviation from planarity of 0.128 Å, and a C20/O5/O6 carboxylate to C17/O3/O4 carboxylic acid twist of 18.6 (2)°.The molecular structure of 4-AcO-MALT hydrofumarate, (II)N,N-diallyltryptammonium (C18H23N2O2+) cation, one half of a fumarate (C2HO2−) dianion, and one half of a fumaric acid (C2H4O2) molecule. The indole ring system of the compound is near planar with a r.m.s. deviation from planarity of 0.016 Å. The ethylammonium arm is turned significantly away from the plane of the indole ring, with a C10—C9—C11—C12 torsion angle of 104.3 (2)°. The complete fumarate dianion is generated through crystallographic inversion symmetry, and is also near planar, with an r.m.s. deviation from planarity of 0.004 Å. The full disordered (vide infra) fumaric acid molecule is generated through inversion, and also demonstrates near planarity, with r.m.s. deviations from planarity of 0.082 and 0.083 Å for the two conformations. One of the allyl groups in the cation is disordered over two orientations with a 0.90 (1):0.10 (1) ratio. The fumaric acid molecule is also disordered over two components with a 0.52 (4):0.48 (4) ratio. The 4-acetoxy group also shows a disorder over two orientations with a 0.62 (4):0.38 (4) ratio. The carboxylate group of the fumarate anion is delocalized, with C—O distances of 1.251 (3) and 1.258 (2) Å.The molecular structure of 4-AcO-DALT fumarate–fumaric acid, (III)N-ethyl-N-methyltryptammonium cations and hydrofumarate anions are linked together in a two-dimensional network lying in the (010) plane through N—H⋯O and O—H⋯O hydrogen bonds (Table 1In the extended structure of (I)s Table 1. The O—Hs Table 1, top. ThN-allyl-N-methyltryptammonium cations and hydrofumarate anions are linked together in an infinite two-dimensional network parallel to (010) through N—H⋯O and O—H⋯O hydrogen bonds (Table 2In the extended structure of (II)s Table 2. The O—Hs Table 2, center.N,N-diallyltryptammonium cations, fumarate dianions and fumaric acid molecules are linked together in a three-dimensional network through N—H⋯O and O—H⋯O hydrogen bonds (Table 3In the extended structure of (III)s Table 3. The O—Hs Table 3, bottom.bet al., 2019aN,N,N-trimethyltryptamine, has been reported . A commercial sample of 4-acetoxy-N-allyl-N-methyltryptammonium hydrofumarate (The Indole Shop) was recrystallized by the slow evaporation of an aqueous solution to yield samples suitable for single crystal X-ray diffraction studies. Single crystals of bis fumarate fumaric acid suitable for X-ray analysis were obtained from the slow evaporation of an acetone solution of a commercial sample (The Indole Shop).Single crystals of 4-acetoxy-Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O). C-bound H atoms were positioned geometrically and refined using a riding model: C—H = 0.95 Å with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989021000116/hb7959sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989021000116/hb7959Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021000116/hb7959Isup5.cmlSupporting information file. DOI: 10.1107/S2056989021000116/hb7959IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989021000116/hb7959IIsup6.cmlSupporting information file. DOI: 10.1107/S2056989021000116/hb7959IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989021000116/hb7959IIIsup7.cmlSupporting information file. DOI: 2053845, 2053844, 2053843CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Nature Communications 10.1038/s41467-020-15822-8, published online 27 April 2020.Correction to: In the original version of this Article “5,5′-dithiobis-(2-nitrobenzoic acid)” was incorrectly written as “5,5′-dithiobis-(2-nitrobenzoic) acid”, and in Eq. (1) the upper limit of the summation in the denominator “n” was incorrectly written as “T”.This has been corrected in both the PDF and HTML versions of the Article.


Background: Thalassemia is an autosomal genetic disorder, found throughout the world. It is still not treatable and create socio economic problems. In this study, we investigated the prevalence and spectrum features of thalassemia in Yunnan Province, the southwestern area of China. During 2014–2018, a total of 3,539 suspected thalassemia children were detected with α- and β-thalassemia mutations by gap-Polymerase Chain Reaction (PCR) and reverse dot blot (RDB) analysis in Kunming Children's Hospital.Results: Of these patients, 1,130 were diagnosed thalassemia gene carriers with a carrying rate of 31.92%. Among them, α-thalassemia was 43.63%, β-thalassemia was 53.98%, cases with both α- and β- thalassemia was 2.39%. In α-thalassemia patients, the most common mutations was –SEA/αα (52.13%), followed by –α3.7/αα (27.79%), hemoglobin H disease (18.46%), and –α4.2/αα (1.62%). Fifteen gene mutations and 30 genotypes were identified in β-thalassemia patients, with the five most common mutations CD17 (A>T) (29.51%), CD41–42 (–TTCT) (27.87%), IVS-II-654 (C>T) (14.92%), CD26 (G>A) (6.89%), and CD26/CD27 (2.62%) accounting for 81.81% of the β-globin gene mutations. Furthermore, we founded two rare mutations CD34 (TGG → TAG) and Int in Chinese populations.Conclusions: Our results suggested that the prevalence and gene mutation spectrum of thalassemia display obviously heterogeneity among children in Yunnan Province. The findings provide the valuable information for premarital and pre-pregnancy screening, prenatal diagnostic services, and designing appropriate prevention programs to control thalassemia for future in this area. Thalassemia is an inherited autosomal recessive disease resulting from mutations in the α- and β-globin gene clusters on chromosome 16 and chromosome 11, respectively. It is characterized by the absence or reduced synthesis of globin chains of hemoglobin and includes two main types, α- and β- thalassemia . It is rHigh prevalence of thalassemia was mainly reported in southern China, Southeast Asia, India, the Middle East, Africa and Mediterranean region . HoweverA total of 3,539 children who were suspected of thalassemia were recruited and tested  from January 2014 to December 2018 in Kunming Children's Hospital, Yunnan, China. The study has been approved by the Ethic Committee of Kunming Children's Hospital. Consent forms were signed by the parents.Peripheral venous blood (5 mL) was collected into an EDTA anticoagulated tube. Peripheral blood counts and red blood cell indices were determined using the automatic blood cell analyzer .The children with low mean corpuscular volume (MCV) values (<82 fl) or low mean cell hemoglobin (MCH) values (<27 pg) were considered possible carriers of thalassemia .Hemoglobin electrophoresis was used to analyze the percent of hemoglobin Hbs A, A2, F, and any abnormal hemoglobin variant. The children with low HbA2 (<2.5%) values were considered possibly α-thalassemia carriers, with high HbA2 (>3.5%) values were considered possibly β-thalassemia carriers, respectively .SEA/αα, –α3.7/αα, –α4.2/αα) were determined by the gap-polymerase chain reaction (Gap-PCR) technique. 17 known β-globin mutations was detected by PCR-reverse dot blot (PCR-RDB) assay. In order to detect unknown and rare β-globin mutations, β-globin gene of all the samples was directly sequenced.According to the instructions . We usedDuring 2014 to 2018, 1,130 of the 3,539 suspected children were diagnosed thalassemia gene carriers with a carrying rate of 31.92% . Among them, α-thalassemia children were 43.63% , β-thalassemia children were 53.98% , children with both α- and β-thalassemia were 2.36% . Three α-globin gene deletion and five genotypes were detected in α-thalassemia children. Fifteen gene mutations and 30 genotypes were identified in β-thalassemia children –3.SEA/αα deletion was the most prevalent genotype, accounting for over one half of the mutations (52.13%), followed by –α3.7/αα (27.79%), hemoglobin H disease (18.46%), and –α4.2/αα (1.62%). Meanwhile, five α-globin genotypes were detected, including 402 children (81.54%) with heterozygous mutations and 91 children (18.46%) with compound heterozygous mutations. Overall, the first three mutations accounted for 98.38% of all α-globin gene mutations.13.93%  children were diagnosed as being carriers of α-thalassemia , three k17.24%  children were carriers of the β-thalassemia, as shown in SEA/αα, CD17/N (14.81%); –SEA/–α3.7, CD26/N (14.81%); –α3.7/αα, CD17/N (11.11%); –SEA/αα, CD41–42/N (11.11%).The distributions of mutations and hematological features are shown in SEA/αα was the most frequent genotype in α-thalassemia and CD17 was the most frequent genotype in β-thalassemia.In the present study, we have investigated the prevalence and mutation characteristics of thalassemia among the children in Yunnan province, the southwestern of China. Among 3,539 suspected children, 1,130 were diagnosed with thalassemia, 493 had α-thalassemia, 610 had β-thalassemia, and 27 were carriers with both α- and β-thalassemia. –It is reported that about 7% of the world's population are carriers of a globin gene mutation . Our stuSEA/αα), right deletion (–α3.7/αα), and the left deletion (–α4.2/αα), which account for 60.81% of thalassemia patients  is the most frequent mutation in Yunnan region (In contrast to α-, β-thalassemia carriers are more often caused by point mutations. At present, more than 300 different β-globin gene mutations have been reported around the world . In Chinn region . The difn region , which wOur data suggested that the prevalence and gene mutation spectrum of thalassemia display obviously heterogeneity among children in Yunnan Province. The findings extended the mutation spectrum of thalassemia in this region, which provided the valuable information for premarital and pre-pregnancy screening, prenatal diagnostic services, and designing appropriate prevention programs to control the incidence of severe thalassemia for future in this area.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by The Kunming Children's Hospital research ethics committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.T-LH, T-YZ, and Y-HY designed the study. XT, Q-LL, C-YS, Y-BL, and YL treated patients and helped draft the manuscript. B-HS, C-HY, and NL performed data collection. T-LH and X-WZ analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Immune checkpoint inhibitor (ICI) monotherapy and combination regimens are being actively pursued as strategies to improve durable response rates in cancer patients. However, the biology surrounding combination therapies is not well understood and may increase the likelihood of immune-mediated adverse events. Accurate stratification of ICI response by non-invasive PET imaging may help ensure safe therapy management across a wide number of cancer phenotypes.18F]AlF-mNOTA-GZP, targeting granzyme B, to stratify ICI response in two syngeneic models of colon cancer, CT26 and MC38. In vivo tumour uptake of [18F]AlF-mNOTA-GZP following ICI monotherapy, or in combination with PD-1 was characterised and correlated with changes in tumour-associated immune cell populations.We have assessed the ability of a fluorine-labelled peptide, [18F]AlF-mNOTA-GZP showed good predictive ability and correlated well with changes in tumour-associated T cells, especially CD8+ T cells; however, overall uptake and response to monotherapy or combination therapies was very different in the CT26 and MC38 tumours, likely due to the immunostimulatory environment imbued by the MSI-high phenotype in MC38 tumours.[18F]AlF-mNOTA-GZP uptake correlates well with changes in CD8+ T cell populations and is able to stratify tumour response to a range of ICIs administered as monotherapies or in combination. However, tracer uptake can be significantly affected by preexisting phenotypic abnormalities potentially confusing data interpretation.[The online version of this article (10.1007/s11307-020-01519-3) contains supplementary material, which is available to authorized users. Thus, combination therapies may act in a complementary or synergistic fashion depending on the tumour microenvironment AlF-mNOTA-GZP, to improve PET imaging sensitivity and spatial resolution [18F]AlF-mNOTA-GZP to stratify immunotherapy response was assessed in response to ICI monotherapy and combined ICI therapies in CT26 and MC38 syngeneic colon cancer models, frequently associated with MSI-low phenotype, and MSI-high phenotype fluoride ion was produced via the [18O18F] nuclear reaction (GE PETtrace 860 cyclotron). Quality control analytical radio-HPLC was performed on a UFLC Shimazdu HPLC system equipped with dual wavelength UV detector and a NaI/PMT-radiodetector . Radioactivity measurements were made with a CRC-55tPET dose calibrator .H-Asp(OtBu)-H NovaSyn TG resin (0.21 mmol/g) was obtained from Merck. Fmoc-amino acids, HATU and HOAt were obtained from Advanced Chemtech. Fmoc-glutamic acid was t-butyl protected. (p-SCN-Bn)-NOTA was purchased from Boc Sciences and Macrocyclics. Glacial acetic acid was purchased from JT Baker. Sep-Pak® light (46 mg) Accell™ plus QMA carbonate cartridges and Sep-Pak® C18 light cartridges were purchased from Waters Corporation. Saline solution  was trapped on a Sep-Pak® light (46 mg) Accell™ plus QMA carbonate cartridge (pre-conditioned with 10 mL water) and washed with a further 5 mL of water. The trapped [18F]fluoride anion was eluted with 0.9 % w/v saline (0.2 mL) and adjusted to pH 4 with glacial acetic acid (1 μL). To the pH-adjusted [18F]fluoride solution was added 2 mM AlCl3 in 0.1 M pH 4 NaOAc buffer (24 μL) and ethanol (0.2 mL). This solution was then added into a reaction vial containing mNOTA-GZP (0.1 mg). The reaction vial was sealed and heated at 100 °C for 15 min without stirring. After cooling to room temperature, the crude reaction mixture was diluted with water (40 mL) and loaded on a Sep-Pak® C18 light cartridge (pre-conditioned with 5 mL ethanol and 10 mL water). The cartridge was washed with a further 5 mL of water. [18F]AlF-mNOTA-GZP was eluted with 70 % ethanol in water (0.4 mL), and diluted with 0.9 % w/v saline to a final concentration of 10 % ethanol in saline. The radiochemical purity of [18F]AlF-mNOTA-GZP was assessed by analytical radio-HPLC (Luna Phenomenex C18(2), 5 μm, 100 Å, 250 mm × 4.6, 0.05 M NH4OAc pH 4.5 (solvent A) and acetonitrile (solvent B), gradient elution 0–6 min 10 % to 95 % B then 6–11 min 95 % B, flow rate 1 mL/min, column temperature 30 °C, λ = 254 nm). The retention time of mNOTA-GZP and [18F]AlF-mNOTA-GZP was between 7.1 to 7.2 min. [18F]AlF-mNOTA-GZP was obtained as a colourless solution in 10 % ethanol in saline (pH = 7.4) with a non-decay corrected radiochemical yield of 17–25 % , with a radiochemical purity of 98–99 % and molar activity 45–90 GBq/μmol (n = 6).NOTA–β-Ala–Gly–Gly–Ile–Glu–Phe–Asp–CHO (mNOTA-GZP) was synthesized using standard Fmoc chemistry and characterized by HPLC and mass spectroscopy as previously described  were prepared in a 1:1 (v/v) ratio in Matrigel (Sigma) and injected subcutaneously into the right shoulder of Balb/c mice or C57BL/6 mice, respectively. Rat IgG2a anti-mouse PD-1 (⍺PD1 mAb RMP1–14), mouse IgG2b anti-mouse CTLA-4 (⍺CTLA4 mAb 9D9), rat IgG1 anti-mouse OX40 (⍺OX40 mAb OX-86), rat IgG2a anti-mouse TIM-3 (⍺TIM3 mAb RMT3–23), rat IgG1 anti-mouse LAG-3 (⍺LAG3 mAb C9B7W), and rat IgG2a isotype control (⍺-trinitrophenol mAb), were purchased from Bio-X Cell. All mice were treated by intraperitoneal (i.p.) injection of either control IgG (5 mg/kg) or monotherapy (⍺PD1 (10 mg/kg), ⍺CTLA4 (5 mg/kg), ⍺OX40 (5 mg/kg), ⍺TIM3 (5 mg/kg) or ⍺LAG3 (10 mg/kg)) or combination therapy (⍺CTLA4 (5 mg/kg), ⍺OX40 (5 mg/kg), ⍺TIM3 (5 mg/kg) or ⍺LAG3 (10 mg/kg) in combination with ⍺PD1 (10 mg/kg)) on days 6, 9 and 12 following tumour inoculation (in order to maintain treatment efficacy ⍺OX40 injected 24 h before ⍺PD1 when used in combination). In vivo subcutaneous tumour lengths were measured by callipers on days 5, 8, 12, and 14 after tumour inoculation. Tumour volume was then calculated using the modified ellipsoid formula 1/2(length × width2) AlF-mNOTA-GZP (~ 10 MBq) via the lateral tail vein. Static PET acquisitions were performed at 60–80 min post-injection (p.i.) and CT and MRI scans were used for co-registration. Animals were monitored for maintenance of body temperature and respiration rate during imaging studies using the Biovet physiological monitoring system. Post-analysis of reconstructed calibrated images were performed with FIJI and Amide software (version 10.3 Sourceforge). Uptake of radioactivity in the tumour was determined by the placement of a volume of interest (VOI) delineated by CT and MR imaging. A VOI was also placed in the quadriceps muscle to provide reference tissue values. Data are expressed as % of the injected dose per gram (%ID/g) of tumour tissue in the VOI.Animals were imaged 14 days after treatment initiation using a Siemens Inveon PET-CT and a Mediso nanoscan 3 T MR-PET. Briefly, animals were anaesthetised using inhalational isoflurane  and injected with AlF-mNOTA-GZP was efficiently prepared by adaptation of the peptide labelling method described by McBride et al. in 2009 [18F]fluoride, [18F]AlF-mNOTA-GZP was obtained in a non-decay-corrected radiochemical yield of 17–25 % as a formulated product suitable for injection  in a total reaction time of 50 min with a radiochemical purity of 98–99 % and molar activity 45–90 GBq/μmol. Similarly, [19F]AlF-mNOTA-GZP reference material was prepared using a similar synthetic procedure  or an ICI combination regime  as shown in Fig. 18F]AlF-mNOTA-GZP PET imaging showed adequate tumour-to-background contrast for tumour visualisation in the two tumour models assessed. [18F]AlF-mNOTA-GZP showed heterogeneous in vivo tumour tracer uptake across the different treatment arms in each of the tumour models  or control treatment in the CT26-tumour bearing mice  and TNRs (0.19 ± 0.06 %ID/g) as well as in the treatment groups that did not inhibit tumour growth . However, significantly higher uptake was observed in treatment groups that successfully inhibited tumour growth including ⍺PD1 alone , ⍺CTLA4 alone , combined ⍺PD1 + ⍺CTLA4 , ⍺OX40 alone , and combined ⍺PD1 + ⍺OX40 .Low [18F]AlF-mNOTA-GZP reliably differentiated between responders and non-responders or control treatment in the MC38 tumour-bearing animals (Table Likewise imaging with 8FAlF-mNOls Table .18F]AlF-mNOTA-GZP uptake was observed in the MC38 tumours. Higher background uptake was observed in the control antibody treated group (0.62 ± 0.14%ID/g) and TNRs (0.77 ± 0.06%ID/g) compared to CT26 tumours. However, even greater uptake was observed in the MC38 tumours in treatment groups that responded to therapy including ⍺PD1 alone , ⍺CTLA4 alone , combined ⍺PD1 + ⍺CTLA4 therapy , ⍺OX40 alone , combined ⍺PD1 + ⍺OX40 therapy , ⍺TIM3 alone , combined ⍺PD1 + ⍺TIM3 therapy , ⍺LAG3 monotherapy  and combined ⍺PD1 + ⍺LAG3 therapy . Response evaluation across all treatment arms showed that [18F]AlF-mNOTA-GZP was significantly higher for responders than non-responders in CT26 tumours    Fig.  and MC3801) Fig. .To quantitate the changes in CT26 TILs in response to therapy, we analysed the distribution of different immune cell markers Table . SignifiP < 0.05), ⍺CTLA4 monotherapy  and ⍺OX40 monotherapy  as well as ⍺PD1 + ⍺CTLA4 combination therapy  and ⍺PD1 + ⍺OX40 combination therapy  compared to control treatment (28.6 ± 4.4) and TNRs (29.4 ± 3.1); however, no significant increases were observed in ⍺TIM3 monotherapy (35.4 ± 6.9), ⍺LAG3 monotherapy (39.3 ± 11.0), ⍺PD1 + ⍺TIM3 combination therapy (34.7 ± 1.9) or ⍺PD1 + ⍺LAG3 combination therapy .Significant increases in CD3+ infiltration was observed in responders to ⍺PD1 monotherapy , ⍺CTLA4 monotherapy  and ⍺OX40 monotherapy  as well as ⍺PD1 + ⍺CTLA4 combination therapy  and ⍺PD1 + ⍺OX40 combination therapy  compared to control treatment (10.4 ± 1.0) and TNRs (11.4 ± 0.9). Again, no significant increases were observed in ⍺TIM3 monotherapy (19.0 ± 3.6), ⍺LAG3 monotherapy (20.6 ± 7.3), ⍺PD1 + ⍺TIM3 combination therapy (19.9 ± 5.0) or ⍺PD1 + ⍺LAG3 combination therapy .Significant increases in CD8+ infiltration were observed in responders to ⍺PD1 monotherapy , ⍺CTLA4 monotherapy  and ⍺OX40 monotherapy  as well as ⍺PD1 + ⍺CTLA4 combination therapy  and ⍺PD1 + ⍺OX40 combination therapy  compared to control treatment (12.1 ± 2.4) or TNRs (11.8 ± 1.3). No significant differences were observed in ⍺TIM3 monotherapy (9.7 ± 2.8), ⍺LAG3 monotherapy (12.9 ± 3.0), ⍺PD1 + ⍺TIM3 combination therapy (13.9 ± 6.7) or ⍺PD1 + ⍺LAG3 combination therapy .Significant increases in CD8+ GZB+ cell infiltration were also observed in responders to ⍺PD1 monotherapy , and CD8+ T cells . The strongest correlation was observed with GZB+ CD8+ T cells .Correlation analysis of Ga-mNOTA-GZP has been shown to be well correlated with immunotherapy efficacy [18F]AlF to improve PET imaging duration of use, sensitivity and spatial resolution [18F]AlF did not substantially change the enzymatic activity of the peptide  response to ⍺PD1 monotherapy and an improved (~ 70 %) response to ⍺CTLA4 or ⍺OX40 monotherapy or combined ⍺PD1 with ⍺CTLA4 or ⍺PD1 with ⍺OX40 therapy, as determined by tumour growth inhibition. The newer combination therapies ⍺TIM3 and ⍺LAG3, however, did not effectively inhibit tumour growth in the CT26 tumour bearing mice when used as monotherapies. Furthermore, neither ⍺TIM3 or ⍺LAG3, effectively inhibited tumour growth when used in combination with ⍺PD1, despite ⍺PD1 monotherapy proving efficacious in 40 % of tumours when used as a monotherapy. This lack of response may be due to the relatively low total number of CT26 resident cytotoxic T cells, despite tumour-infiltrating CD8+ cells having been shown to express TIM-3 and LAG-3 [18F]AlF-mNOTA-GZP at ~ 0.2%ID/g in non-responsive tumours, peaking at ~ 0.6%ID/g in treatment responsive tumours. The MC38 tumours, with a much greater number of cytotoxic T cells, displayed a higher response rate to all therapies . [18F]AlF-mNOTA-GZP uptake was similarly affected by the immunostimulatory environment, with significantly higher uptake in all the MC38 tumours  ranging from ~ 0.6%ID/g in non-responsive tumours (levels comparable to peak responders in the CT26 tumour type) and up to ~ 1.1%ID/g in treatment-responsive tumours. In both tumour types, uptake of [18F]AlF-mNOTA-GZP in TRs were positively correlated with CD3+, CD8+ and CD8+GZB+ TILs  occurs in approximately 15 % of colorectal cancers  and rendnd LAG-3 , 30. Themodest ~ 0 % respoFurthermore, while the number of non-responsive MC38 tumours was relatively low, those that did not respond displayed an intriguing reduction in CD8+ TILs compared with the control treated group, potentially linked with the regulatory suppression mechanisms that caused these tumours to be non-responsive. While a small increase in CD4+ FOXP3+ CD25+ Tregs was observed in the MC38 TNRs when compared to the control treated tumours this was not significant. Certainly, the presence of mutations can influence the function of several cellular processes and these may include those that are designed to nullify the host response.in vivo molecular imaging of intra-tumoural granzyme B using [18F]AlF-mNOTA-GZP is able to stratify tumour response to a range of ICIs administered to preclinical colon cancer models given as monotherapies or in combination. However, care should be taken in interpreting the images, as tracer uptake can be significantly affected by pre-existing phenotypic abnormalities. Further studies will be needed to elucidate other factors affecting the interpretation of granzyme B targeting tracers before they could be used for stratification of patient response across multiple tumour phenotypes.In summary, the current study shows that ESM 1(PNG 71 kb)High Resolution Image (TIF 163 kb)ESM 2(PNG 379 kb)High Resolution Image (TIF 731 kb)ESM 3(PNG 49 kb)High Resolution Image (TIF 155 kb)ESM 4(PNG 1306 kb)High Resolution Image (TIF 5663 kb)ESM 5( 31 kb)


The mean Zn—N(pyrrole) bond length is 2.058 Å and the Zn—O(diazoester) bond length is 2.179 (4) Å. The zinc cation is displaced by 0.2202 (13) Å from the N4C20 mean plane of the porphyrinate anion toward the O atom; the involvement of this atom leads to a [100] polymeric chain in the crystal.In the title compound, [Zn(C Remarkably little variance in these bond lengths occurs, even with varied functionality around the diazo moiety Fig. 2et al., 20165-[4-(Hydroxymethyl)phenyl]-10,15,20-(4-(trimethoxy)phenyl)porphyrin and the zinc starting complex were synthesized using previously reported methods (Carrie 2Cl2 solution (5 ml) of zinc 5-[4-(hydroxymethyl)phenyl]-10,15,20-triphenylporphyrin , 3 eq. of 1,8-diazabicyclo[5.4.0]undec-7-ene  were first added under argon at 273 K and then 1.5 eq. of bromo acetyl bromide (0.2 mmol). The reaction mixture was stirred for 10 min at room temperature. After cooling the solution again to 273 K, a THF solution of ditosylhydrazine (2 eq.) and DBU (5 eq.) was added, and the mixture was stirred for 30 min at room temperature. The solution was then evaporated, dissolved in CH2Cl2 and purified through a silica gel column (CH2Cl2). Yield = 70%. Red prisms of (I)2Cl2): lmax, nm: 421, 548, 592.To a distilled CHPLATON  = 1.2Ueq(C) or 1.5Ueq(methyl C). The methyl H atoms were allowed to rotate, but not to tip, to best fit the electron density. The crystal studied was refined as an inversion twin.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989020001085/hb7871sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020001085/hb7871Isup2.hklStructure factors: contains datablock(s) I. DOI: 1968288CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: