Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non‐fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low‐affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4‐amino‐phenyl‐acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.Discovery of protein‐binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide  Site‐directed screening? Formylglycine (red) is a reactive amino acid that can be utilized in fragment ligation assays. Incorporated into a site‐directing hexapeptide it can effectively probe the active site of PTP1B, yielding new phosphotyrosine mimetics derived from different nucleophiles, such as aryl amines (green). DK values to enable detection.Site‐directed discovery of small protein‐binding fragments with M<250 Da is a challenge due to the low affinities of most fragments and due to the lack of analytical methods that enable the detection of binding fragments at precisely defined positions on the protein surface.Fragment ligation has been introduced as a method to enhance binding of a primary fragment by a covalent, typically protein‐templated reaction with a secondary ligand.We reasoned that peptides and proteins carrying a reactive side chain electrophile should enable the site‐specific ligation of nucleophiles and thus might serve as tools for the site‐directed discovery of binding fragments for defined protein pockets Figure . FormylgThese findings encouraged us to investigate formylglycine peptides for the site‐directed discovery of protein‐binding fragments. As a model system, we selected the active sites of protein tyrosine phosphatases (PTP) which recognize and hydrolyze phosphotyrosine residues  as substrates.1 (X=O) or the potent inhibitor 2 derived from it (X=CF2) and replaced the phosphotyrosine residue by formylglycine  1 contains the autophosphorylation sequence 988–993 of epidermal growth factor receptor (EGF−R) including the O‐phosphorylated tyrosine 992  and has been recognized as a substrate of the phosphotyrosine phosphatase PTP1B with a MK value of 3.6 μM ‐4 was synthesized following a published 7‐step protocol from L‐serine,4 instead and established a facilitated access to 4 in only 3 steps starting from O‐ethyl 2‐nitro‐acetate 5. Treatment of 5 with titanium tetrachloride, N,N‐diisopropylethylamine, and triethyl orthoformate afforded the diethyl acetal 6 (conditions a).6 in the presence of Raney nickel furnished ethyl 2‐amino‐3,3‐diethoxypropionate 7. Alternatively, under conditions b, N‐formylglycine ethylester 8 was formylated in C2 position with ethyl formate and tBuOK, followed by acetalization with HCl in EtOH at −25 °C and cleavage of the N‐formyl group under basic conditions leading to the same diethylacetal 7.7 followed by N‐Fmoc protection with Fmoc‐OSu afforded the desired N‐Fmoc‐formylglycine 4  in a total yield of 68 % over 4 steps  or 43 % over 3 steps .The phospho‐hexapeptide Ac‐Asp‐Ala‐Asp‐Glu‐Tyr*‐Leu‐NH4 was then used in solid phase peptide synthesis on polystyrene with Rink amide linker by diisopropyl carbodiimide/HOBt activation. Cleavage and complete deprotection was achieved with TFA/H2O. (95 : 5 v/v) and the formylglycine peptide 3 was isolated by HPLC in 73 % yield. Peptide 3 was highly soluble in water, buffer, and DMSO, so that we were able to analyze its structure and reactivity in solution .Building block 3 synthesized from (S)‐fGly and rac‐fGly were identical. In DMSO the fGly‐residue appears as a mixture of the aldehyde form 3, the hydrate form 9, and the enol form 10. In H2O/D2O 9 : 1, exclusively the hydrate form 9 was observed with both diastereomeric Hα at 4.3 ppm and both Hβ at 5.3 ppm. In the DQF‐H,H‐COSY spectrum all backbone NH protons were detectable suggesting that no cyclization with any of the backbone amides occurred and the formyl moiety thus was available for ligation. High resolution Q‐TOF MS analysis further confirmed the predominant hydration of the formylglycine peptide 3.NMR‐spectra of 3 were investigated with several nucleophilic fragments F detected in a primary screening experiment (see below) using NMR spectroscopy . One can suspect that the Z‐configuration of 11 is strongly favored over the E‐configuration due to an H‐bond between the enamine‐NH and the carbonyl residue of Glu4 resulting in a 7‐membered ring . Formation of the ligation product 11 was followed in Watergate 1H NMR for 1000 min by integration of the benzylic CH2 group in starting material F1 and product 11 and indicated product formation with an average half reaction time of 10.7 min ‐trifluoromethyl sulfonamide F2 yielded enamine 12  formed hydrazone 13 with 100 % conversion of 3 instantaneously (<2 min). 13 was characterized by a shifted Hα of the fGly residue at 5.0 coupling with the Hβ at 7.2 and the fGly‐NH at 8.3 ppm. A single configuration of the hydrazone double bond was formed, presumably the thermodynamically favored E‐isomer. One might suspect as the preferred conformation the one establishing an H‐bonded, 6‐membered ring between the double‐bonded hydrazine‐N1 and the NH of Leu4  (1 : 5), a common reducing agent in biochemistry and especially in assays with protein tyrosine phosphatases, yielded a ligation product detectable in HPLC‐MS with a mass corresponding to the hemi‐thioacetal 14 . 14 could not be detected in NMR or isolated, which is in full agreement with earlier results on the transient formation of bioactive hemi‐(thio)‐acetals formed by protein‐templated ligation reactions.3 DTT was found to compete with other nucleophiles such as amines or hydrazines. Therefore, it had to be replaced in enzyme assays with a non‐nucleophilic reducing agent. No ligation reaction was observed between formylglycine peptide 3 and tris‐carboxyethyl phosphane (TCEP) which was found to sustain the enzymatic activity of protein tyrosine phosphatases reliably at 50 μM for several hours and therefore was employed in all ligation experiments.Incubation of 3 was tested as an inhibitor of two protein tyrosine phosphatases, PTP1B and SHP2, in an enzyme activity assay using DiFMUP  as a fluorogenic substrate. Peptide 3 bound to and inhibited PTP1B with a IK value of 484 μM and SHP2 with a IK of 341 μM  were selected and tested as inhibitors of PTP1B alone and in combination with peptide 3 . Fragments were pre‐selected for library composition based upon representation of potential phosphate‐mimetic substructures including carboxylic acids, sulfonic acids, sulfonamides, and fluorine‐rich functional groups, which were to be investigated for fluorine‐specific interactions with phosphotyrosine binding sites., 687.2711 Da. calcd m/z 688.2790 [M+H]+, 710.2609 [M+Na]+, found m/z 688.2792 [M+H]+, 710.2607 [M+Na]+.3,3‐Diethoxy‐2‐(9H‐fluoren‐9‐ylmethoxycarbonylamino)‐ propanoic acid 4: Ethyl 2‐amino‐3,3‐diethoxypropanoate 7  was suspended in 0.5 M aq. LiOH solution (5 mL) and methanol was added until a clear solution remained. It was stirred at room temperature for 2 h. TLC control after this time revealed the hydrolysis of 7 after which the mixture was neutralized with Amberlite® IR‐120 loaded with H+. The resin was filtered off and washed with water and concentrated under reduced pressure. The product of hydrolysis and NaHCO3  were stirred in H2O (10 mL) at 0 °C. To this solution was added dropwise 9‐fluorenylmethyl N‐succinimidyl carbonate  dissolved in 1,4‐dioxane (10 mL) at 0 °C. The mixture was stirred for 1 h at 0 °C, warmed to room temperature and then stirred for another 12 h. The reaction mixture was concentrated under reduced pressure and purified by column chromatography to provide 4 as white solid ; Rf=0,46 . 1H NMR: : δ=7.75 , 7.61 , 7.39 , 7.31 , 5.67 , 4.85 , 4.59 , 4.39 , 4.24 , 3.88–3.77 , 3.76–3.6 , 3.65–3.57 , 1.32 , 1.21  ppm. 13C NMR: : δ=172.33 (COOH), 156.59 (CONH), 143.80 (Ar−Cquart), 141.37 (Ar−Cquart), 127.83 (Ar−C), 127.19 (Ar−C), 125.28 (Ar−C), 120.06 (Ar−C), 101.17 (Cβ), 67.53 (Fmoc CH2), 64.74 (CH2CH3), 64.69 (CH2CH3), 56.73 (Cα), 47.15 (Fmoc CH), 15.20 (CH2CH3), 15.08 (CH2CH3) ppm. HRMS: (ESI): C22H25NO6 [M], 399.1682 Da. calcd m/z 398.1604 [M−H]−, 422.158 [M+Na]+, found m/z 398.1609 [M+H]+, 422.1578 [M+Na]+.Ethyl 3,3‐diethoxy‐2‐nitropropanoate 6: To a solution of ethyl‐nitroacetate 5  in anhydrous methylene dichloride (25 mL) held at ‐ 10 °C under an argon atmosphere was slowly added by syringe titanium(IV)‐chloride . The mixture was stirred for 10 min and N,N‐diisopropylethylamine  was added to the mixture dropwise over 30 min. The Mixture was held at −10 °C with stirring for 1 h. triethyl‐orthoformate  was added to the mixture dropwise and stirring was continued for 2 h at −10 °C. The reaction mixture was diluted with a 20 % solution of ethanol in saturated aqueous NaHCO3 (100 mL) and the mixture was stirred vigorously for 10 min. Organic solvents were removed from the mixture under reduced pressure. Water (200 mL) was added to the concentrated reaction mixture and the aqueous phase was extracted three times with ethylacetate. The combined organic phases were filtered over celite and dried over Na2SO4. Evaporation of the solvents under reduced pressure afforded compound 6 as yellow oil ; Rf=0,46 . 1H NMR: : δ=5.24 , 5.17 , 4.28 , 3.81–3.75 , 3.69–3.61 , 1.30 , 1.19  ppm. 13C NMR: : δ=161.77 (COOEt), 100.08 (Cβ), 88.52 (Cα), 64.79 (OCH2CH3), 64.60 (OCH2CH3), 63.18 (COOCH2CH3), 15.16 (OCH2CH3), 15.07 (OCH2CH3), 13.94 (COOCH2CH3) ppm. HRMS: C9H17NO6 [M], 235.1056 Da. calcd m/z 258.0948 [M+Na]+, 274.0687 [M+K]+, found m/z 258.0948 [M+Na]+, 274.0680 [M+K]+.Ethyl 2‐amino‐3,3‐diethoxypropanoate 7 (Method A): Ethyl 3,3‐diethoxy‐2‐nitropropanoate 6  was dissolved in absolute ethanol (5 mL) and hydrogen was bubbled through the mixture for 5 min. raney nickel was added and the mixture was hydrogenated at atmospheric pressure for 12 h at room temperature. The reaction mixture was filtered through celite, and the filter cake was washed plentiful with ethanol. Filtrate was concentrated under reduced pressure, leaving a reddish oil. Vacuum distillation of this afforded 7 as yellowish oil ; Rf=0,29 . 1H NMR: : δ=4.61 , 4.24–4.16 , 3.77–3.67 , 3.61 , 3.58–3.51 , 2.05–1.95  1.30–1.25 , 1.23–1.19 , 1.19–1.15  ppm. 13C NMR: : δ=161.8 (COOEt), 100.1 (Cβ), 88.5 (Cα), 64.8 (OC2CH3), 64.60 (OC2CH3), 63.2 (COOC2CH3), 15.2 (OC2CH3), 15.1 (OC2CH3), 13.9 (COOC2CH3) ppm. HRMS: (ESI): C9H19NO4 [M], 205.1314 Da. calcd m/z 206.1392 [M+H]+, 228.1206 [M+Na]+, found m/z 206.1379 [M+H]+, 228.1195 [M+Na]+.Ethyl 2‐amino‐3,3‐diethoxypropanoate 7 (Method B): A solution of N‐formylglycine ethylester 8  in ethylformate  was added dropwise to a mixture of potassium tert‐butoxide  in toluene (60 mL) at 10 °C over a periode of 2 h. Stirring was continued for additional 2 h after which the mixture was allowed to stand at 4 °C for 18 h. The supernatant was discarded and the gelantinious residue dissolved in ethanol (35 mL). This solution was the diluted with methylene dichloride (50 mL) and cooled to −25 °C and treated with HCl gas for 3 h. The mixture was then stirred for 24 h at room temperature. The solution was concentrated under reduced pressure and the residue was suspended in diethyl ether (80 mL). This suspension was treated with saturated K2CO3 until strongly basic when the phases were separated, and the organic phase was washed further with water and dried over Na2SO4 and evaporated under reduced pressure to afford an oil. Vacuum distillation of this afforded 8 as yellowish oil .NMR ligation experiments: The ligation of peptide 3 with fragments F1, F2 or F3 was confirmed by NMR experiments. The respective fragments were dissolved in a solution of 3, to yield a sample with a final concentration of 10 mM of the fragment and 5 mM of 3. Experiments were performed in 9 : 1 H2O/D2O or buffer  at 300 K. Experiments were performed using a WATERGATE water suppression.Peptide 15: Formylglycine peptide 3  and amine F1  were stirred in dry MeOH (1 mL) and AcOH (20 μL) with molecular sieve  for 1 h at room temperature. NaCNBH3 (2.5 equiv) was added and the mixtures was stirred for 18 h. Molecular sieves were filtered off and the mixture was concentrated under reduced pressure. The residue was purified by RP flash column chromatography yielding 15 as a white solid ; HRMS: (ESI): C35H50N8O15 [M], 822.3396 Da. calcd m/z 823.3474 [M+H]+, 821.3317 [M−H]−, found m/z 823.3477 [M+H]+, 821.3316 [M−H]−.Peptide 16: Following the procedure for peptide 15, 3  and F4  yielded 16 as white solid ; HRMS: (ESI): C35H50N8O16 [M], 838.3345 Da. calcd m/z 839.3423 [M+H]+, 837.3267 [M−H]−, found m/z [M+H]+, [M−H]−.Peptide 17: Following the procedure for peptide 15, 3  and barbituric acid  yielded 17 as white solid ; HRMS: (ESI): C31H45N9O16 [M], 799.2984 Da. calcd m/z 800.3063 [M+H]+, 798.2906 [M−H]−, found m/z 800.3058 [M+H]+, 798.2912 [M−H]−.Peptide 18: Following the procedure for peptide 3, using the unnatural amino acid 19, 18 was obtained as a white solid  from 200 mg resin. 1H NMR: : δ=8.21 , 8.16 , 8.06 , 8.03 , 7.97 , 7.78 , 7.23 , 7.04 , 6.98 , 6.64 , 4.59–4.54 , 4.54–4.50 , 4.44 , 4.30–4.24 , 4.26–4.24 , 4.21 , 4.18 , 3.34 , 3.23 , 2.77–2.69 , 2.69–2.66 , 2.59–2.55 , 2.26–2.19 , 1.97–1.89 , 1.85 , 1.81–1.73 , 1.63–1.56 , 1.51–1.45 , 1.22 , 0.87 , 0.82 . HRMS: (ESI): C34H48F3N9O15S [M], 911.2943 Da. calcd m/z 912.3021 [M+H]+, 934.2840 [M+Na]+, found m/z 912.3021 [M+H]+, 934.2838 [M+Na]+.2‐N‐(9H‐Fluoren‐9‐ylmethoxycarbonylamino)‐3‐(4‐trifluoromethyl‐sulfonamido)‐phenyl)‐amino‐propanoic acid 19: To a solution of 20  and NaHCO3  in water (10 mL) was added N‐(9‐fluorenylmethoxy‐carbonyloxy)‐succinimide  dissolved in 1,4‐dioxane (10 mL) dropwise at 0 °C. The mixture was allowed to slowly warm up to room temperature and was subsequently stirred for 18 h. Water was added (25 mL) and the mixture extracted with ethyl acetate (3×50 mL). The organic layer was washed with 0.2 M HCl (40 mL) and brine (40 mL) and dried over Na2SO3. The solvents were removed under reduced pressure and the crude product purified via MPLC yielding 19 as a brown solid . 1H NMR:  δ=7.90 , 7.72 , 7.68 , 7.42 , 7.33 , 7.31 , 6.99 , 6.63 , 4.35–4.29 , 4.24 , 4.23–4.20 , 3.42 , 3.36 . 13C NMR:  δ=172.88 (COOH), 156.62 (CONH), 147.93 (Ar−Cquart), 144.25 (Fmoc Ar−Cquart), 141.18 (Fmoc Ar−Cquart), 128.09 (Fmoc Ar−C), 127.54 (Fmoc Ar−C), 127.17 (Fmoc Ar−C), 125.68 (Fmoc Ar−C), 122.78 (Ar−Cquart), 121.26, 120.57 (Ar−C), 119.42 (CF3), 112.83 (Ar−C), 66.17 (Fmoc CH), 53.75 (Cα), 47.08 (Fmoc CH2), 44.52 (Cβ). HRMS: (ESI): C25H22F3N3O6S [M], 549.1181 Da. calcd m/z 550.1260 [M+H]+, 572.1079 [M+Na]+, found m/z 550.1260 [M+H]+, 572.1080 [M+Na]+.2‐Amino‐3‐(4‐trifluoromethyl‐sulfonamido‐phenyl)‐amino‐propanoic acid 20: A mixture of L‐2‐N‐Boc‐2,3‐diamino‐propanoic acid (Boc‐Dap‐OH) , 1‐fluoro‐4‐nitrobenzene  and K2CO3  in ethanol (15 mL) was stirred for 24 h at 90 °C. To the crude residue of this reaction was added 10 % Pd/C and the flask wash flushed with H2 and left under H2 atmosphere for 18 h at room temperature. The catalyst was filtered off, the solvent evaporated under vacuum and purified by column chromatography to provide N‐2‐Boc‐3‐(4‐aminophenyl)‐2,3‐diamino‐propanoic acid as brownish solid . 1H NMR:  δ=6.41 , 6.33 , 6.08  3.64 , 3.11 , 2.94 , 1.38 . 13C NMR:  δ=172.95 (COOH), 155.55 (Boc CO), 141.09 (Ar−Cquart), 139.52 (Ar−Cquart), 116.07 (Ar−C), 114.29 (Ar−C), 78.08 (Boc Cquart), 54.97 (Cα), 48.36 (Cβ), 28.79 (Boc CH3). HRMS: (ESI): C14H21N3O4 [M], 295.1532 Da. calcd m/z 296.1610 [M+H]+, 294.1454 [M−H]−, found m/z 296.1612 [M+H]+, 294.1451 [M−H]−. The obtained intermediate  was dissolved in anhydrous DCM (10 mL) under an argon atmosphere and the reaction mixture was cooled to 0 °C. Trifluoromethanesulfonic anhydride  was added dropwise to the reaction mixture and stirred for 30 min at 0 °C, after which the mixture was slowly warmed to room temperature and stirred further for 18 h. The reaction was diluted with water (20 mL), filtered and the feed was washed twice with MeCN/H2O . The solvents were evaporated under vacuum and purified by column chromatography to provide 20 as brownish solid . 1H NMR:  δ=7.20 , 6.86 , 6.77 , 6.45 , 3.53 , 3.42 , 3.22 . 13C NMR:  δ=174.37 (C‐2), 143.13 (C‐5), 125.16 (C‐6), 123.58 (C‐8), 118.43 (C‐10), 113.56 (C‐7), 53.77 (C‐1), 45.69 (C‐3). HRMS: (ESI): C10H12F3N3O4S [M], 327.0501 Da. calcd m/z 328.0579 [M+H]+, 326.0422 [M−H]−, found m/z 328.0582 [M+H]+, 326.0419 [M−H]−.Z)‐2‐‐5‐oxo‐1,5‐dihydro‐4H‐pyrazol‐4‐ylidene)‐hydrazinyl)‐phenyl)‐acetic acid (21)(: A solution of NaNO2 (0.17 mmol) in H2O (100 μL) was added at 0 °C to a suspension of 2‐(4‐aminophenyl)‐acetic acid (0.17 mmol) in 4 M HCl (0.3 mL). The acidic solution was stirred for 1 h at 0 °C, after which a solution of 2,5‐bis(4‐nitrophenyl)‐2,4‐dihydro‐3H‐pyrazol‐3‐one (0.15 mmol) in THF (2 mL) was added and the mixtures was stirred for 10 min at 0 °C. Afterwards a solution of 1.3 M NH4OH (0.2 mL) was added, stirred for 1 h at 0 °C and for 24 h at r.t. The suspension was filtered under suction, the solid was washed with H2O (2×20 mL), and dried in air. A mixture of EtOAc:DCM:n‐Hex  was added in a sealed flask for 2 h. The suspension was filtered off, washed with Et2O and dried under vacuo to provide product 21. 1H NMR: : δ=8.55 , 8.51 , 8.34 , 8.29 , 7.54 , 7.29 , 3.56  ppm. 13C NMR: : δ=173.6 (COOH) 157.9 (C‐7), 147.1 (Pyr Cquart), 146.3 (Ar−Cquart), 143.88 (Pyr Cquart), 142.4 (Ar−Cquart), 141.3 (Ar−Cquart), 130.4 (Ar−C), 130.0 (Ar−Cquart), 129.1(Ar−C), 125.4 (Ar−C), 123.9 (Ar−C), 122.3 (Ar−Cquart), 121.2 (Ar−Cquart), 120.9 (Ar−C), 117.6 (Ar−C), 32.0 (CH2) ppm. HRMS: (ESI): C23H16N6O7 [M], 488.1080 Da. calcd m/z 489.1159 [M+H]+, 487.1002 [M−H]−, found m/z 489.1162 [M+H]+, 487.1005 [M−H]−. Anal: calcd for C22H14F3N7O7S: C, 56.56; H, 3.30; N, 17.21; found: C, 57.12; H, 3.58; N, 17.56.(Z)‐4‐‐5‐oxo‐1,5‐dihydro‐4H‐pyrazol‐4‐ylidene)‐hydrazinyl)‐phenyl)‐1,1,1‐trifluoromethanesulfonamide (22): Following the procedure for compound 21, using N‐(4‐aminophenyl)‐1,1,1‐trifluoromethanesulfonamide  and 2,5‐bis(4‐nitrophenyl)‐2,4‐dihydro‐3H‐pyrazol‐3‐one , 22 was obtained as an orange solid . HRMS: (ESI): C22H14F3N7O7S [M], 577.0628 Da. calcd m/z 578.0706 [M+H]+, found m/z 578.0696 [M+H]+. Anal: calcd for C22H14F3N7O7S: C, 45.76; H, 2.44; N, 16.98; found: C, 45.78; H, 2.54; N, 16.98.Z)‐2‐‐5‐oxo‐1,5‐dihydro‐4H‐pyrazol‐4‐ylidene)‐hydrazinyl)‐phenoxy)‐acetic acid (23)‐acetic acid  and 2,5‐bis(4‐nitrophenyl)‐2,4‐dihydro‐3H‐pyrazol‐3‐one , 23 was obtained as an orange solid . HRMS: (ESI): C23H16N6O8 [M], 504.1030 Da. calcd m/z 505.1108 [M+H]+, 503.0951 [M−H]−, found m/z 505.1106 [M+H]+, 503.0976 [M−H]−. Anal: calcd for C23H16N6O8: C, 54.77; H, 3.20; N, 16.66; found: C, 55.23; H, 3.18; N, 15.62.Protein Expression and purification: DNA encoding the catalytic domain of human PTP1B (amino acids 1–321) was subcloned into the pQLinkH vector. The gene encoding the N‐terminal His7‐tagged protein was over‐expressed at 17 °C in E. coli Rosetta (DE3). The purification procedure comprises an affinity chromatography on a 5 mL HisTrap FF crude column , charged with Ni2+, and a size‐exclusion chromatography on a Superdex 26/60 column  equilibrated with 25 mM HEPES‐NaOH (pH=7.5), 50 mM NaCl, 1 mM DTT. The His7‐tag was cleaved with tobacco etch virus protease at 20 °C prior to the gel‐filtration step. The catalytic domain of human SHP2 (amino acids 225–541) was purified from E. coli B21 (DE3). The overexpressed protein was fused to an N‐terminal His10‐tag. Cells were harvested and resuspended in 25 mM Tris‐HCl (pH=7.5), 50 mM NaCl, 1 % (v/v) Triton X‐100, 10 % (v/v) glycerol, 1 mM DTT. After cell lysis, debris was removed by centrifugation at 12,000 rpm for 30 min. The supernatant was filtered prior to affinity purification with Ni‐NTA agarose (Qiagen). The slurry was washed with 50 mM Tris‐HCl (pH=8.0), 500 mM NaCl supplemented with 25 mM imidazole. Reasonably pure protein was eluted with 200–300 mM imidazole. The purification was completed by size‐exclusion chromatography with a Superdex 75 16/600 column  equilibrated with 20 mM HEPES‐NaOH (pH=7.5), 50 mM NaCl, 1 mM DTT.Enzyme activity assays of SHP2 and PTP1B: The catalytic activity of SHP2 catalytic domain and PTP1B were monitored using the fluorogenic substrate DiFMUP . Phosphatase reactions were performed at room temperature in 384‐well black plate, clear flat bottom, low flange, non‐binding surface  using a final volume of 20 μL and the following assay buffer conditions: 50 mM MOPS, pH=6.5, 200 mM NaCl, 50 μM TCEP, 0.03 % Tween‐20 (freshly added prior to each measurement). Test compounds were dissolved in DMSO or buffer at stock concentrations of 50, 20 or 10 mM and serially diluted. Enzyme  and different concentrations of the tested compounds were incubated in buffer for 30–60 min at room temperature. Measurements were performed with a final concentration of 2.5 % DMSO unless stated otherwise at 37 °C and were performed in triplicate. Enzymatic reactions were started by adding DiFMUP  concentrations matching the experimentally determined MK values of the enzymes of 67 μM (PTP1B) or 72 μM (SHP2). Samples were excited at a wavelength of 360 nm and emitted fluorescence was recorded at 460 for 10 min using a microplate reader . Initial slope of fluorescence was determined in triplicate and 50IC values were calculated with GraphPad Prism 5. Determined 50IC values were converted into the corresponding IK values applying the Cheng Prusoff equation IK=50IC/(1+[S]/MK ).Dynamic ligation screening of nucleophilic fragments with 3: Under assay conditions described above, PTP1B or SHP2 were pre‐incubated with 3 (100 μM) for 5 min. Subsequently, (200 μM) of the respective fragments were added and the resulting mixtures were incubated for 30 min at RT, prior to DiFMUP addition.Determination of apparentKI‐values of 11 in a time‐dependent assay: Under the conditions described above, PTP1B was pre‐incubated with peptide 3 in different concentrations  for 5 mins. Subsequently, over the course of 60 mins (every 15 min), 2 equivalents of fragment F1 were added to each of the different concentrations of 3 at RT, prior to DiFMUP addition.Determination of apparentKI‐values of 12 in a concentration‐dependent assay: Under the conditions described above, PTP1B was pre‐incubated with peptide 3 at different concentrations  for 5 min. Subsequently, fragment F2 was added at three different concentrations  and the resulting mixtures were incubated for 30 min at RT, prior to DiFMUP addition.Computational Methods: The protein X‐ray diffraction crystal structure of mutated PTP1B (PDB code: 1PTU)3, 9, 11, 12, 15, 16, and 18 were docked to the binding pocket of PTP1B using Schrödinger's GLIDE.Cellular experiments: HeLa cells were cultivated in DMEM buffer with 10 % FCS in 75 cm2 cell culture flasks at 37 °C, 5 % CO2. At confluency of about 75 %, cells were seeded in a density of 250,000 cells/mL in 6‐well plates and incubated for 24 h. Subsequently, medium was removed and replaced by DMEM buffer with 0.1 % BSA (1 mL per well) and incubated for another 16 h. Hepatocyte growth factor  and test compounds in different final concentrations  were added. Control wells received the same amount of DMSO  or no addition. Plates were incubated for 1 h, then washed with cold PBS and shaken with lysis buffer  for 5 min. Cell lysates were transferred to Eppendorf cups and centrifuged at 4 °C with 10,000 g for 10 min. Total protein was quantified in cell lysates using RotiNanoquant (Carl Roth # K880.1) and 30 μg of protein was applied to 12 %‐SDS‐PAGE with a run time of 90 min at 150 V. Then protein was blotted in Towbin bufer to an PVDF‐membrane over 1 h at 100 V, and the membrane was saturated with 20 ml TBS‐Tween/ 2 % BSA, 1 h at RT. The blot was incubated with anti‐phospo‐ERK as primary antibody  overnight at 4 °C, washed 3×5 min with TBS‐Tween, and incubated with anti‐mouse IgG‐hrp as secondary antibody . The blot was again washed 3×5 min with TBS‐Tween and imaged with the ECL system (ThermoScientific # 34080) at Syngene PXi Imager. From the same lysis sample, under the same conditions, ERK 1/2 and β‐tubulin were blotted. For β‐tubulin, the antibody incubation time was adjusted to 2 h at RT.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The chains are interdigitated.The nearly planar molecule is centrosymmetric with two all- 70H112N6O6, is centrosymmetric with two all-s-trans chains, the other two chains have an s-cis unit starting with the oxygen atoms. The chains are interdigitated in the packing.The nearly planar molecule of the title compound, C The centrosymmetric molecule is located parallel to the (110) plane, the five aromatic rings are almost coplanar with torsion angles of −0.4 (4)° (N6—C5—C3—C2) and −2.0 (4)° (C11—C10—C8—N7). Two dodecyloxy chains per molecule are completely all-s-trans organized whereas the other pair shows an s-cis-conformation of the O16—C17—C18—C19 unit. Torsion angles at the all-trans chains are 179.3 (2) for C13—C14—O29—C30, 177.9 (5)° for C14—O29—C30—C31 and 178.1 (2)° for O29—C30—C31—C32 but for the other chain, 15.8 (3)° (C17—O16—C12—C13), 176.2 (2)° (C12—O16—C17—C18), and −55.3 (3)° for the O16—C17—C18—C19 unit. The packing is controlled by interaction of the aliphatic chains  is located above the pyrazine centroid.Electron-deficient conjugated oligomers are interesting as electron-transporting materials in organic electronics : 2916, 2848, 1602, 1544, 1468, 1442, 1429, 1174; 1H NMR , 6.66 , 4.04 , 3.17 , 1.82 , 1.55-1.42 , 1.42-1.20 , 0.93-0.83 . Whereas the absorption spectrum of the title compound in cyclohexane peaks at 348 nm, three maxima appear in methanol , indicating aggregation. The emission of a solution in cyclohexane is centered at 403 nm, and in toluene at 409 nm. Crystallization was via slow evaporation of a solution in chloroform/2-propanol.The title compound was prepared Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314623000342/bt4131sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314623000342/bt4131Isup2.hklStructure factors: contains datablock(s) I. DOI: 2235881CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit contains three independent molecules differing slightly in conformation. Portions of the observed conformations are determined by intramolecular N—H⋯O hydrogen bonds. In the crystal, O—H⋯O hydrogen bonds form chains of molecules which are linked into corrugated sheets parallel to ( 14H13NO4, contains three independent molecules, which differ slightly in conformation. Each contains an intramolecular N—H⋯O hydrogen bond. In the crystal, O—H⋯O hydrogen bonds form chains of molecules, which are linked into corrugated sheets parallel to (The asymmetric unit of the title compound, C These compare quite favorably with those found in molecules with R = Me  differ modestly in the orientations of the methyl groups while the third differs more in conformation from the other two Fig. 1. In each3.B⋯O7, O5—H5B⋯O11 and O9—H9B⋯O3 hydrogen bonds repeating in that order  Å, C10⋯Cg2 = 3.731 (2) Å, C10=O2⋯Cg2 = 91.41 (13)°  Å, C24⋯Cg6 = 3.694 (2) Å, C24=O6⋯Cg6 = 91.12 (14)° ; O10⋯Cg4 = 3.4110 (18) Å, C38⋯Cg4 = 3.656 (2) Å, C38=O10⋯Cg4 = 91.00 (13)° ]. The layers are held together by C11—H11C⋯O6 hydrogen bonds  are smaller than 1.0% with low densities of points.In order to visualize the intermolecular interactions, a Hirshfeld surface (HS) analysis  using the standard B3LYP functional and 6–311 G basis-set calculations , hardness (η), potential (μ), electrophilicity (ω) and softness (σ) are recorded in Table 3E)-3-[1-(2-hydroxyphenylamino)ethylidene]-6-methyl-3H-pyran-2,4-dione ring. The energy band gap [ΔE = ELUMO − EHOMO] of the molecule is 4.54 eV, and the frontier molecular orbital energies, EHOMO and ELUMO are −6.12 and −1.58 eV, respectively.The optimized structure of the title compound in the gas phase was generated theoretically 6.Gaussview software  was used to broadly predict reactive sites for electrophilic and nucleophilic attack in the title compound by B3LYP/6-31G optimized geometries using 7.et al., 2016A  yielded 66 hits of which 15 were deemed most similar to the title molecule. These include molecules with R = Me  in 30 mL of ethanol, 2.5 mmol of dehydroacetic acid were added. The mixture was refluxed for 1 h. After cooling, the precipitate that formed was recrystallized from ethanol solution to give yellow crystals in 88% yield.9.Uiso(H) = 1.2Ueq or 1.5Ueq.Crystal, data collection and refinement details are presented in Table 410.1107/S2056989022007514/zn2020sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022007514/zn2020Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022007514/zn2020Isup3.cmlSupporting information file. DOI: 2192044CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N‐Acetylneuraminic acid  is one of a large, diverse family of nine‐carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac2, as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards. We report here the synthesis of 9‐O‐ and 4‐O‐acetylated sialic acids  with optimisation of previously reported synthetic routes. Neu5,9Ac2 was synthesised in 1 step in 68 % yield. Neu4,5Ac2 was synthesised in 4 steps in 39 % overall yield. Synthesis was followed by analysis of these standards via quantitative NMR (qNMR) spectroscopy. Their utilisation for the identification and quantification of specific acetylated sialic acid derivatives in biological samples is also demonstrated. Synthesised acetylated derivatives of N‐acetylneuraminic acid, Neu5,9Ac2 and Neu4,5Ac2, alongside commercially available Neu5Ac and Neu5Gc were utilised as standards for the quantitative analysis of these derivatives in plasma and serum samples. Multiple derivatives could be detected in one assay, which exhibited high specificity and low limits of detection and quantitation. Neu5Ac is a nine‐carbon backbone monosaccharide with a carboxylic acid functional group Figure  and is oN‐glycans as the terminating unit  was treated with trimethyl orthoacetate in the presence of catalytic p‐toluene sulfonic acid for 20 minutes to afford Neu5,9Ac2(2) in 68 % yield after purification using ion‐exchange chromatography  to 3.95 and 4.26 ppm in (2).Neu5,9Ac, Neu5Ac  was trea2(3) was based on work by Ogura et al.(3) therefore commenced with protection of the carboxylic acid group to give a methyl ester using conditions developed by Malapelle et al. employing methanol and trifluoroacetic acid to give (4) in quantitative yield.+ resin to give (5) in 65 % yield. Deprotection of the methyl ester is required in the next step. The method put forth by Ogura et al. using 1 M NaOH was utilised to give (6) in quantitative yield. The acetyl group was installed at the 4‐position using excess pyridine and acetic anhydride and the crude mixture was then purified using ion exchange chromatography using 1 M formic acid as the eluent. Removal of the formic acid under reduced pressure at 35 °C, as opposed to the use of lyophilisation by Ogura et al. then afforded (3) in 60 % yield  to 5.17 ppm in the target compound (3).The route chosen to synthesise Neu4,5Ac2 and Neu4,5Ac2 in hand, the next step was to determine the purity of the standards using qNMR techniques. The method chosen for this was based on the pulse length based concentration determination (PULCON) method,20D−13 ° ; mp. 153–155 °C; 1H NMR : δ 4.26 , 4.07 , 4.00–3.90 , 3.87–3.77 , 3.49 , 2.19 , 2.00 , 1.94 , 1.81‐1.72 : δ 174.73 (C13), 174.41 (C11), 171.22 (C14), 95.48 (C2), 70.15 (C7), 69.2 (C8), 67.59 (C6), 66.80 (C4), 66.26 (C9), 52.03 (C5), 38.89 (C3), 22.6 (C12), 20.19 (C15) ppm; IR vmax [cm−1] (powder) 3295 , 1719 (C=O), 1034 (C−O); HRMS (ESI): m/z calc for C13H22O10N: 352.1238 [M+H]+; found: 351.1239.Methyl 5‐Acetamido‐3,5‐dideoxy‐D‐glycero‐D‐galactononulopyranosonate (4):N‐acetylneuraminic acid 1  in methanol (40 mL) was added TFA . This was stirred at room temperature for 48 hrs. The solvent was removed under reduced pressure to give the methyl ester 4 as a white solid  [α]20D−26 ° ; mp. 177–178 °C; 1H NMR : δ 3.94 , 3.80 , 3.72 , 3.60 , 3.50 , 3.42 , 2.19 , 1.92 , 1.79  ppm 13C NMR : δ 174.79 (C13), 171.36 (C11), 95.29 (C2), 70.29 (C6), 70.05 (C8), 68.14 (C7), 66.62 (C4), 63.10 (C9), 53.43 (C14), 52.00 (C5), 38.60 (C3), 22.00 (C12) ppm; IR vmax [cm−1] (powder) 3256 , 2944 (C−H), 1751 (C=O), 1026 (C−O); HRMS (ESI): m/z calc for C12H21O9NNa: 352.1109 [M+H]+; found: 346.1102.O‐isopropylidene‐D‐glycero‐β‐D‐galactononulopyranosonate (5)Methyl 5‐acetamido, 3,5‐dideoxy‐8,9‐:4  in acetone (50 mL) was added Amberlyst 15 H+ resin (1.5 g) and 2,2‐dimethoxypropane . This was stirred for 3.5 hrs at room temperature before being filtered to remove the resin. The resin was washed with acetone and the filtrate collected. After removing the solvent under reduced pressure, the resulting crude material was passed over a silica plug and eluted with ethyl acetate (4×200 mL) to give acetonide 5 as a beige foam  [α]20D−24 ° ; mp. 164–166 °C; 1H NMR : δ 8.07 , 6.71 , 4.82 , 4.75 , 4.02‐3.94 , 3.90‐3.86 , 3.85‐3.78 , 3.68 , 3.56 , 3.52‐3.49 , 2.03 , 1.88 , 1.61 , 1.26 , 1.23  ppm 13C NMR : δ 179.94 (C13), 171.68 (C11), 107.22 (C15), 94.57 (C2), 75.92 (C8), 71.70 (C7), 68.38 (C6), 65.20 (C9), 64.94 (C4), 52.89 (C14), 52.12 (C5), 40.23 (C3), 26.55 (C16), 25.72 (C16), 22.55 (C12) ppm; IR vmax [cm−1] (powder) 3291 , 2926 (C−H), 1740 (C=O), 1032 (C−O); HRMS (ESI): m/z calc for C15H25O9NNa: 386.1422 [M+Na]+; found: 386.1416.O‐isopropylidene‐D‐glycero‐D‐galactononulopyranosonate (6)5‐Acetamido‐3,5‐dideoxy‐8,9‐:5  was dissolved in 1 M NaOH (5 mL) and stirred for 4 hrs at room temperature. This was then diluted with water (10 mL) and deionised with Amberlyst H+ resin. The mixture was filtered, the filtrate was collected and lyophilised to give a white solid 6 . [α]20D−25 ° ; mp. 157–158  °C; 1H NMR : δ 4.25 , 4.20‐4.11 , 4.07‐3.97 , 3.95‐3.85 , 3.62 , 2.09 , 1.46 , 1.36  ppm; 13C NMR : δ 176.75 (C13), 174.43 (C11), 168.30 (C2), 109.58 (C14), 75.10 (C8), 70.59 (C7), 69.07 (C6), 66.99 (C9), 66.14 (C4), 52.16 (C5), 25.77 (C15), 24.27 (C15), 21.99 (C12) ppm; IR vmax [cm−1] (powder) 3282 , 1735 (C=O), 1427 (COOH) 1057 (C−O); HRMS (ESI): m/z calc for C14H23O9NNa: 372.1265 [M+H]+; found: 372.1263.O‐acetyl‐D‐glycero‐D‐galactononulopyranosonate (3)5‐Acetamido‐3,5‐dideoxy‐4‐:6  in dry pyridine (0.5 mL) under nitrogen was added acetic anhydride . This was stirred at room temperature for 18 hours after which ethanol was added to remove excess acetic anhydride and solvent removed under reduced pressure by co‐evaporation with toluene (3×100 mL). The resultant residue was dissolved in water (10 mL) and passed through a column (2×10 cm) of DOWEX‐1X8 formate anion exchange resin (100–200 mesh). The column was washed with water (3×10 mL) and eluted with formic acid (50 mL). The formic acid was removed under reduced pressure to give a clear residue. The residue was dissolved in water (5 mL) and lyophilised to give 4‐O N‐acetylneuraminic acid 3 as a white solid . [α]20D−34° ; mp. 170–172 °C; 1H NMR : 5.22‐5.12 , 4.18‐3.99 , 3.76‐ 3.72 , 3.63 , 3.50 , 2.24 , 1.95 , 1.87 ; 13C NMR : δ 174.7 (C14), 173.38 (C13), 173.31 (C11), 95.24 (C2), 70.07 (C8), 70.00 (C7), 68.01 (C6), 63.09 (C4), 53.50 (C9), 49.38 (C5), 36.02 (C3), 21.85 (C12), 20.30 (C15) ppm; IR vmax [cm−1] (powder) 3340 , 2933 (C−H), 1727 (C=O), 1019 (C−O); HRMS (ESI): m/z calc for C13H22O10N: 352.1238 [M+H]+; found: 351.1239.Quantitative NMR analysis: Into an NMR tube was placed 600 μL of a 5.03 mMol maleic acid solution in D2O. A 1.8 mg quantity of synthesised standard was dissolved in 1.8 mL of D2O. This was split into three 600 μL samples in order to analyse the standards in triplicate. After matching and tuning, each NMR sample was analysed on a 500 MHz NMR spectrometer where a 1D 1H spectrum with water suppression was obtained with a relaxation delay of 20 seconds. The 360 ° pulse was obtained for each spectrum using pulsecal. The pulse sequence used was noesypr1D. Equation 1 was used to determine the concentration and purity of each synthesised standard.2 and Neu4,5Ac2 standardsDMB labelling of the Neu5Ac, Neu5Gc, Neu5,9Ac: Neu5Ac, Neu5Gc, Neu5,9Ac2 and Neu4,5Ac2 standards were labelled using LudgerTagTM DMB Sialic Acid (LT‐KDMB‐96). DMB labelling solution (20 μL) was added to the standards. The samples were then vortexed and centrifuged followed by incubation at 50 °C for 3 hours. The labelling reaction was quenched by the addition of water to give a final volume of 500 μL (480 μL). Standard curves were prepared by performing serial dilution to create a standard curve with points: 0.01, 0.02, 0.1, 0.2, 0.5, 1.0 nmol. The procedure was carried out using a Hamilton MICROLAB STARlet Liquid Handling Robot.Sialic acid release and DMB labelling of human plasma and guinea pig serum: Release of sialic acid and DMB labelling of the samples was achieved using LudgerTagTM DMB Sialic Acid (LT‐KDMB‐96). A 5 μL aliquot of each sample was dispensed into a 96‐well plate in triplicate. To this was added 25 μL of 2 M acetic acid. The sample was vortexed and centrifuged followed by incubation at 80 °C for 2 hours. The sample was allowed to cool to room temperature and a 5 μL aliquot of each sample was transferred to a new 96‐well plate. To this was added 20 μL of DMB labelling solution. The sample was then vortexed and centrifuged followed by incubation at 50 °C for 3 hours. The labelling reaction was quenched by the addition of water to give a final volume of 500 μL (475 μL). The samples were then subjected to a 1 in 10 dilution . All procedures, except the dispensing of the samples on the plate, were conducted using a Hamilton MICROLAB STARlet Liquid Handling Robot.Sialic acid release and DMB labelling of porcine and ovine serum: Release of sialic acid and DMB labelling of the samples was achieved using LudgerTagTM DMB Sialic Acid (LT‐KDMB‐96). A stock solution was first prepared by dissolving 10 mg of porcine or ovine serum in 1 mL ultra‐pure water. A 10 μL aliquot taken from the solution of each sample was dispensed into a 96‐well plate in triplicate. To this was added 25 μL of 2 M acetic acid. The sample was vortexed and centrifuged followed by incubation at 80 °C for 2 hours. The sample was allowed to cool to room temperature and a 5 μL aliquot of each sample was transferred to a new 96‐well plate. To this was added 20 μL of DMB labelling solution. The sample was then vortexed and centrifuged followed by incubation at 50 °C for 3 hours. The labelling reaction was quenched by the addition of water to give a final volume of 500 μL (475 μL). The samples were then subjected to a 1 in 10 dilution . All procedures, except the dispensing of the samples on the plate, were carried out using a Hamilton MICROLAB STARlet Liquid Handling Robot.2Sialic acid release and DMB labelling of plasma for Neu5,9Ac: Release of Sialic Acid and DMB labelling of the samples was achieved using LudgerTagTM DMB Sialic Acid (LT‐KDMB‐96). A 25 μL aliquot of each sample was dispensed into a 96‐well plate in triplicate. To this was added 75 μL of 2.666 M acetic acid. The sample was vortexed and centrifuged followed by incubation at 80 °C for 2 hours. The sample was allowed to cool‐room temperature and a 20 μL aliquot of each sample was transferred to a new 96‐well plate. To this was added 20 μL of DMB labelling solution. The sample was then vortexed and centrifuged followed by incubation at 50 °C for 3 hours. The labelling reaction was quenched by the addition of water to give a final volume of 500 μL (475 μL). The sample was subjected to filtration through a Ludger Clean Protein Binding Membrane filtration plate (LC‐PBM‐plate) to remove excess protein. All procedures, except the dispensing of the samples on the plate and LC‐PBM‐plate filtration, were carried out using a Hamilton MICROLAB STARlet Liquid Handling Robot.Fluorescence analysis of DMB labelled sialic acid derivatives by LC‐fluorescence detection: DMB labelled sialic acid derivatives were analysed by LC‐FLD. A 5 μL aliquot of each sample was injected to a LudgerSep™ uR2 UHPLC column  at 30 °C on a Dionex UltiMate™3000 RSLCnano system with a fluorescent detector . For Neu5Ac analysis, an isocratic solvent system was used (7 : 9 : 84 MeOH : ACN : H2O) for 15 minutes including an ACN wash. For Neu5,9Ac2 analysis a variable solvent system was used: 7 : 6 : 87 MeOH:ACN:H2O for 6.5 minutes followed by 6 : 9 : 85 MeOH:ACN:H2O for 11.5 minutes. Integration of resultant peaks was performed using Chromeleon 7. LOD and LOQ regression analysis and calculation was performed using Microsoft Excel 2019.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The bridging O2− ligand is located on a twofold rotation axis, resulting in point group 2 for the entire complex. The MnIII atom is displaced out of the 24-atom mean plane of the porphyrine entity by 0.52 Å. C—H⋯π and π–π interactions help to stabilize the molecular packing within the crystal structure.In the crystal structure of the title oxido-bridged binuclear complex, [Mn The two MnIII ions are displaced by 0.48 and 0.52 Å from their respective 24-atom mean plane. It is interesting to note that the μ-oxido species [Mn(OEP)]2O is very unstable in halocarbon solvents . Figs. 12O are bridged by a single oxido ligand with an Mn—O distance of 1.7600 (3) Å and an Mn—O—Mn bridging angle of 176.1 (2)°. The Mn1⋯Mn1′ separation 2(OH)}ClO4, the title compound shows virtually the same metal displacement from the 24-atom mean plane (0.52 Å), while a larger Mn—O—Mn bridging angle [176.1 (2) versus. 160.4 (8)°] and a shorter Mn—O distance [1.7600 (3) versus. 2.026 (1) Å] is observed.In the crystal structure of the title complex, the asymmetric unit contains one deprotoanted porphyrin molecule located in general position and an oxygen atom on a twofold rotation axis ]Cl were prepared according to literature protocols ]2O (10 mg) was dissolved in 4 ml of chlorobenzene and cannula-transferred into 8 mm glass tubes, then carefully layered with hexanes before sealing the tubes. X-ray quality crystals were obtained several weeks later.The title compound was prepared following a reported procedure  I. DOI: 10.1107/S2414314622008690/wm4172Isup2.hklStructure factors: contains datablock(s) I. DOI: 2204345CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Longer N—H⋯O hydrogen bonds link the tetramers into [010] chains.In the title hydrated Schiff base, the dihedral angle between the aromatic rings is 5.06 (11)° and an intramolecular O—H⋯N hydrogen bond closes an S(6) ring. In the crystal, O 13H12N4O3·H2O, the dihedral angle between the aromatic rings is 5.06 (11)° and an intramolecular O—H⋯N hydrogen bond closes an S(6) ring. In the crystal, Ow—H⋯O and Ow—H⋯N (w = water) hydrogen bonds link the components into centrosymmetric tetramers (two Schiff bases and two water molecules). Longer N—H⋯O hydrogen bonds link the tetramers into [010] chains. A weak C—H⋯O hydrogen bond and aromatic π–π stacking between the pyrazine and phenyl rings [centroid–centroid separations = 3.604 (2) and 3.715 (2) Å] are also observed.In the title hydrated Schiff base, C Their applications include molecular switches (Coskun S(6) ring. The C7—N2 bond length [1.278 (3) Å] is consistent with a normal carbon–nitrogen double bond. In the crystal, Ow—H⋯O and Ow—H⋯N (w = water) hydrogen bonds link the components into centrosymmetric tetramers (two Schiff base and two water molecules). Longer N—H⋯O hydrogen bonds link the tetramers into [010] chains ° and an intramolecular O2—H2⋯N2 hydrogen bond closes an s Table 1. The pacPyrazine-2-carbohydrazide  was reacted with 2-hydroxy-3-methoxybenzaldehyde  under reflux in 25 ml methanol for 8 h. After cooling and solvent removal by rotary evaporation, a light yellow solid was obtained, which was recrystallized from methanol solution at room temperature to obtain colourless crystals of the title compound.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314619017310/hb4335sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314619017310/hb4335Isup2.hklStructure factors: contains datablock(s) I. DOI: 1973543CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Orientogalba ollula has been reported as intermediate hosts of many zoonotic trematodes. Here, we investigated the prevalence of zoonotic trematodes within O. ollula in Guangxi, China, and assessed their zoonotic potential.Food-borne parasitic diseases decrease food safety and threaten public health. The snail species is an intermediate host for numerous human parasitic trematodes. Echinostoma revolutum was recorded by dissecting infected ducklings from 1-day post infection (dpi) to 10 dpi.Snails were collected from 54 sites in 9 cities throughout Guangxi. The snail and trematode larvae species were determined by combining morphological characteristics and molecular markers. The trematodes prevalence and constituent ratio were calculated and compared among different habitat environments. Phylogenetic trees of the trematode species were constructed using the neighbor-joining method with nuclear internal transcribed spacer 2 (ITS2) sequences. The developmental cycles of the isolated trematodes were examined by experimental infection in ducks. The developmental characteristics of O. ollula from 11 sample sites. Morphological together with molecular identification, showed that E. revolutum, Australapatemon sp., Hypoderaeum conoideum, Pharyngostomum cordatum, and Echinostoma sp. parasitized O. ollula, with the highest infection rate of E. revolutum (13.0%). However, no Fasciola larvae were detected. The trematodes prevalence and constituent ratio varied in two sub-biotypes (P < 0.01). A neighbor-joining tree analysis of ITS2 sequences resulted in distinct monophyletic clades supported by sequences from isolated larvae with high bootstrap values. Ducklings exposed to O. ollula infected with Echinostoma sp., E. revolutum, and H. conoideum larvae were successfully infected. The animal model for Echinostoma revolutum was successfully established. E. revolutum matured from larvae to adult at 10 dpi in the intestine of the duck, and the developmental characteristics of E. revolutum were characterized by the maturation of the reproductive and digestive organs at 6–8 dpi.The overall prevalence of trematode larvae was 22.1% (1818/8238) in O. ollula from Guangxi, China. Existing trematodes infection in animals and human clinical cases, coupled with the wide geographical distribution of O. ollula, necessitate further evaluations of the potential risk of spillover of zoonotic infection from animal to human and vice versa.This study revealed a high prevalence of zoonotic trematodes in The online version contains supplementary material available at 10.1186/s40249-022-01014-7. Corresponding trematodes belong to the families Fasciolidae, Paragonimidae and Echinostomatidae  in the feces, and the morphological characteristics of adult Echinostoma sp. were presented as measurements: body length (9.8 ± 1.8 mm), width (1.2 ± 2.3 mm), oral sucker [(638.9 ± 1.9) × (399.2 ± 15.8) μm], acetabulum [(1591.2 ± 17.8) × (1338.2 ± 19.6) μm], pharynx [(492.6 ± 54.3) × (331.7 ± 74.8) μm], anterior testis [(1120.5 ± 139.6) × (707.4 ± 49.2) μm], posterior testis [(1274.9 ± 181.1) × (880.4 ± 49.2) μm], and ovary [(818.9 ± 10.2) × (527.9 ± 48.3) μm].We conducted an infection experiment for three kinds of trematode to evaluate the rates of parasite establishment in ducklings. Ducklings were individually exposed to H. conoideum eggs in ducklings fed with infected O. ollula from three sites after a median duration of 12 dpi (range: 9 dpi to 14 dpi). The morphological characteristics of adult H. conoideum were as follows: body length (1.05 ± 0.61 mm), width (1.5 ± 0.27 mm), oral sucker [(424.2 ± 16.1) × (293 ± 11.2) μm], acetabulum [(1610.2 ± 98.4) × (1594.6 ± 198.7) μm], pharynx [(379.8 ± 53.2) × (253.6 ± 44.9) μm], anterior testis [(1902.6 ± 164.6) × (875.3 ± 140.8) μm], posterior testis [(2045.2 ± 255.8) × (898.2 ± 139.2) μm], ovary [(751.7 ± 129.3) × (553.9 ± 110.6) μm], and the possession of 50 spines. E. revolutum eggs [(104.1 ± 15.1) × (63.1 ± 12.6 μm] were found on 10 dpi. The morphology of the adult E. revolutum was characterized by: body length (8.5 ± 1.2 mm), width (2.2 ± 0.7 mm), oral sucker [(260.0 ± 32.5) × (180.1 ± 19.8) μm], acetabulum [(741.6 ± 23.7) × (598.3 ± 25.1) μm], pharynx [(193.1 ± 30.5) × (150.6 ± 27.8) μm], anterior testis [(628.0 ± 31.2) × (459.4 ± 27.8) μm], posterior testis (725.5 ± 17.1 × 557.9 ± 18.7 μm), ovary [(411.8 ± 18.5) × (311.2 ± 15.4) μm], and the presence of a head collar with 37 spines.In contrast, we found E. revolutum in duckling hosts, because there were insufficient snails infected with metacercariae of Echinostoma sp. and H. conoideum. The developmental characteristics of E. revolutum were recorded by dissecting infected ducklings from 1 to 10 dpi when eggs in the feces were first detected. E. revolutum could be obtained in the small intestine from 1 to 7 dpi and then migrate and reside in the cecum and colon from 8 to 10 dpi. The body length developed from 490 μm to 8500.5 μm (a 17-fold increase). At 1 dpi, juveniles had a circumoral collar bearing 37 spines in a double circle and characterized by clearly visible oral suckers, acetabulum, pharynx, esophagus, and cecum. At 1 dpi, the tiny structure of the testis appeared. By 4 dpi, the ovaries were beginning to organize and develop, and the seminal receptacle began to form. The tubular-shaped uterus loomed at 4 dpi, and maturation of the reproductive and digestive organs occurred at 6 to 8 dpi. The vitelline glands were the last to appear, and several eggs deposited in the uterus were observed at 9 dpi. E. revolutum larvae matured at 10 dpi and excreted eggs .


Aquatrifluoridoboron and ethylene carbonate form a 1:2 co-crystal with a C=O⋯H—O—H⋯O=C hydrogen-bonding motif. 3H2O·2OC(OCH2)2, was determined by low-temperature single-crystal X-ray diffraction. The co-crystal crystallizes in the orthorhombic space group P212121 with four formula units per unit cell. The asymmetric unit consists of an aquatrifluoridoboron molecule and two ethylene carbonate molecules, connected by O—H⋯O=C hydrogen bonds. This crystal structure is an interesting example of a superacidic BF3H2O species co-crystallized with an organic carbonate.The crystal structure of the co-crystal of aquatrifluoridoboron with two ethylene carbonate  molecules, BF The overall shape of the BF3 moiety in BF3H2O in terms of bond lengths and angles is similar to that of the BF4− anion  graph-set motif 2 co-crystal were discovered when a crystalline sample of the air-sensitive BF3·OC(OCH2)2 adduct was examined under a protective cold nitrogen stream at about −50 °C. The BF3·OC(OCH2)2 compound was synthesized from dry ethylene carbonate and BF3 gas under anhydrous conditions, as described previously 2 were located in a droplet at the tip of the aluminium trough  I. DOI: 10.1107/S2414314623000627/wm4182Isup2.hklStructure factors: contains datablock(s) I. DOI: 2237804CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


D2 symmetry. They show a largely twisted double bond, the molecular halves enclosing dihedral angles of 62.86 (4) and 61.22 (3)°.The asymmetric unit of the title compound contains two almost identical molecules, which have approximate  42H24, contains two almost identical molecules. The molecules have approximate D2 symmetry. They show a largely twisted double bond, the molecular halves enclosing dihedral angles of 62.86 (4) and 61.22 (3)°. The crystal studied was twinned by non-merohedry.The asymmetric unit of the title compound, C The dihedral angle between these planes is 62.86 (4)° and the length of the central C1A—C22A bond is 1.408 (5) Å. The values for molecule B are very similar: maximum deviations from the mean planes are −0.293 (4) Å for C5 (B1) and 0.170 (4) Å for C26 (B2). The dihedral angle amounts to 61.22 (3)° and the length of the central C1B—C22B bond is 1.395 (5) Å. Whereas the length of the twisted and elongated bond connecting the two halves of molecules A and B  correlates very well with the calculated value of 1.408 Å, the calculated torsion angle of 52° is about 10° too small. The solid material gives a very weak single ESR signal.Benzoannulated fulvenes attract chemists because of their electronic properties  I, global. DOI: 10.1107/S2414314622001699/bt4120Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622001699/bt4120Isup3.cmlSupporting information file. DOI: 2151512CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The different intra and intermolecular hydrogen-bonding interactions in the crystal structure of the title salt are discussed. 11H17N2O+·C7H3N2O7−, exhibits secondary nitrogen atoms (N—H) in the 2-methoxyphenylpiperazine (2MeOPP) cation, which is protonated with a phenolic hydrogen atom of 3,5-dinitrosalicylic acid (DNSA). One of the oxygen atoms of the nitro group in the 3,5-dinitrosalicylate anion is disordered over two orientations with occupancy factors of 0.65 (7) and 0.35 (7) . The 2-methoxyphenylpiperazinium cation and 3,5-dinitrosalicylate anion are linked in the asymmetric unit by a bifurcated N—H⋯O hydrogen bond, which formed is between the H atom in the protonated piperazinium unit of the cation and the carboxylic acid group in the anion. The piperazine ring adopts a chair conformation. The crystal structure features N—H⋯O and C—H⋯O hydrogen bonds interactions, which lead to the formation of a sandwich-like arrangement. Hirshfeld surface analysis was used to determine the relative contributions of various intermolecular interactions, indicating that that H⋯O/O⋯H (38. 3%) and H⋯H (31. 8%) contacts are the major contributors.The title salt , C Piperazine and substituted piperazine derivatives are often used as intermediates for a wide range of pharmaceuticals, polymers, dyes, corrosion inhibitors and surfactants. In particular, (2-methoxyphenyl)piperazine derivatives are used as 5-HT1A receptor ligands with reduced α1-adrenergic activity -tartaric acid] salts and their supramolecular features have been reported piperazine is a substituted cyclo aliphatic amine with two nitrogen atoms at opposite positions of the six-membered ring. A substituent 2-methoxyphenyl group is attached to one of the nitrogen atoms while the other has one attached hydrogen atom , 3,5-dinitro-2-oxidobenzoate and piperazine, we have now investigated the crystal structure of 1-(2-methoxyphenyl) piperazinium 3,5-dinitrosalicylate (I)2.Pī with the asymmetric unit comprising one 2-methoxyphenylpiperazinium (2MeOPP)1+ cation and one 3,5-dinitrosalicylate (DNSA)1− anion  Å, θ = 176.3 (3)°, φ = 338 (4)°. One of the oxygen atoms of the nitro group (atom O4) in the 3,5-dinitrosalicylate anion is disordered over two orientations with occupancy factors of 0.65 (7) and 0.35 (7). Both nitro groups, the phenoxylate oxygen atom and a carboxylic acid group in the anion are coplanar with an r.m.s. deviation of 0.0074 Å. A bifurcated intermolecular N—H⋯O hydrogen bond [N3—H3A⋯O5 = 2.936 (3) Å and N3—H3A⋯O6 = 3.153 (3) Å] links the cation and anion in the asymmetric unit.The title salt crystallizes in the triclinic space group on Fig. 1. The pipKα COOH = 2.2) is easier than that of the phenolic –OH group (pKα OH = 6.8). 62 carboxylate moiety structures (COO−) and 70 phenolate anion structures (O−) were found in a search of the Cambridge Structural Database  Å, which also indicates that the strong intramolecular hydrogen bond between the O6 and O7 atoms. Similar types of intramolecular hydrogen bonds were observed in salicylic acid with a distance of 2.62 Å  interaction, which links two neighbouring cations and anions into a centrosymmetric tetrameric architecture, which is further stabilized by the C14—H14⋯O5v interaction [3.481 (3) Å] and yields a macrocyclic ring structure with an ii interaction [3.581 (3) Å], which links two neighbouring (DNSA)1− units with an 1− units are further linked through the previously mentioned bifurcated N3—H3A⋯ interaction and the N3—H3B⋯O7i [2.787 (3) Å], C10—H10A⋯O4A [3.118 (10) Å] interactions into a layered structure propagating parallel to the b axis , and N3—H3B⋯O7], the N3—H3B⋯O7 interaction is stronger [D⋯A = 2.787 (3) Å] than the other two, which is due to the fact that two charged components are involved in this interaction, i.e. the phenolate O7 atom in DNSA−1 and the protonated N3—H3B unit in 2MeOPP+1. All of the above interactions facilitate the arrangement of the DNSA1− ions in a layered molecular structure. The top and bottom sides of the DNSA1− layers are stabilized by the two adjacent cationic layers. As a result, a sandwich-like arrangement is observed. Briefly, the layered DNSA1− units form the core with the top and bottom sides of the cation layers arranged facing. An overall packing diagram is shown Fig. 6The oxygen atoms in both nitro groups (O1–O4), the carboxylic acid group (O5 and O6) and a phenolate moiety (O7) in the DNSA anion all act as acceptors for various intermolecular N—H⋯O and C—H⋯O interactions, except for atom O4 Table 1. In the if Fig. 3. Atom O1if Fig. 4. Neighbois Fig. 5. Of the 4.Crystal Explorer 17.5  are shown in Fig. 7A, O7, O6, H10B and H3B suggest that these atoms participate in hydrogen-bonding interactions (see Table 1de + di = 1.8 Å] make the most significant contribution (38.3%), followed by H⋯H contacts [symmetrical blunt spikes at de + di = 2.4 Å], which contribute 31.8%, while C⋯H, N⋯H, C⋯O, O⋯N, C⋯N and C⋯C contacts contribute 11.6%, 1.7%, 6.7%, 2.7%, 1.9%, 0.5% and 2.8%, respectively. Other significant peaks for various non-covalent contacts are indicated in the FP plot piperazine  and 3,5-dinitrosalicylic acid  in an equimolar ratio. The stoichiometrically (1 mmol) weighed starting materials were completely dissolved in 50 mL of methanol at room temperature and stirred continuously for 3 h. The homogeneous solution was filtered using Whatmann filter paper and placed in a dust-free atmosphere, and allowed to evaporate slowly at room temperature. A suitable single crystal was harvested after a growth period of 25 days.7.Uiso(H) = 1.2Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022006831/ex2058sup1.cifCrystal structure: contains datablock(s) I, publication_text. DOI: 2183987CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Intermolecular N—H⋯O, C—H⋯O and C—H⋯N hydrogen bonds form a three-dimensional network in the crystal, connecting molecules through the O atoms of solvent molecules. 16H11BrN6O·2C2H6OS, the 1,2,3,7-tetrahydroimidazopyridine ring system and the oxindole moiety are both nearly planar  and their planes form a dihedral angle of 86.04 (5)° with each other. Intermolecular N—H⋯O, C—H⋯O and C—H⋯N hydrogen bonds link molecules in the crystal through the O atoms of the solvent molecules, generating a three-dimensional network. A Hirshfeld surface analysis was performed to further analyse the intermolecular interactions.In the title compound, C These ring systems make a dihedral angle of 86.04 (5)° with each other. The cyano (–C≡N) and amine (NH2) groups form an intermolecular hydrogen bond with one dimethyl sulfoxide (DMSO) group, giving an S(10) motif  (see Scheme3.A—N1 bond (which has some multiple-bond character) acts as an electron donor to Br1 in a kind of ‘halogen bond’, with a Br1⋯C8A distance of 3.284 (2) Å.In the crystal, molecules are linked through the O atoms of the DMSO solvent molecules by intermolecular N—H⋯O and C—H⋯O hydrogen bonds which, together with C—H⋯N hydrogen bonds, form a three-dimensional (3D) network Table 1. The π-cCrystalExplorer  plotted over the range from −0.6206 to 1.3180 a.u. The interactions given in Table 1The Hirshfeld surfaces were calculated and the two-dimensional (2D) fingerprint plots generated using a), and those delineated into H⋯H, N⋯H/H⋯N, O⋯H/H⋯O, C⋯H/H⋯C and Br⋯H/H⋯Br contacts are shown in Figs. 4b)–(f). The percentage contributions to the Hirshfeld surfaces from the various interatomic contacts are as follows: H⋯H , N⋯H/H⋯N , O⋯H/H⋯O , C⋯H/H⋯C  and Br⋯H/H⋯Br . Other minor contributions to the Hirshfeld surface are from Br⋯C/C⋯Br (3.9%), Br⋯N/N⋯Br (2.0%), C⋯C (1.5%), S⋯C/C⋯S (0.8%), S⋯H/H⋯S (0.6%), S⋯N/N⋯S (0.4%), O⋯N/N⋯O (0.4%) and Br⋯O/O⋯Br (0.3%).The overall 2D fingerprint plot for (1) is given in Fig. 44.et al., 2016H-indol-2-one unit of (1) gave 87 hits. The three compounds most resembling (1) are (I)   , N—H⋯O hydrogen bonds lead to the formation of chains along the c-axis direction. Within the chains there are further N—H⋯O and C—H⋯O hydrogen bonds enclosing via N—H⋯O and C—H⋯O hydrogen bonds involving the dimethyl sulfoxide solvent molecule which acts as both an acceptor and a donor.In the crystal of (II), the asymmetric unit contains two independent molecules (A and B) having similar conformations. In the crystal, molecules are linked by N—H⋯O hydrogen bonds, forming chains along the a axis which enclose In (III), two intramolecular N—H⋯O hydrogen bonds are formed, each closing an S(6) loop. In the crystal, strong N—H⋯O hydrogen bonds lead to the formation of zigzag chains along the c axis. These are consolidated in the 3D crystal packing by weak N—H⋯O hydrogen bonding, as well as by C—H⋯O, C—H⋯Br and C—H⋯π interactions.In  solution . Single crystals of (1) were grown from DMSO solution.To a solution of 2-(5-bromo-2-oxoindolin-3-ylidene)malononitrile , which was previously prepared by a known procedure : δ 3.50 , 6.61 , 6.78 , 7.35 , 7.37 , 7.73 , 10.44 . 13C NMR : δ 42.46 (CH2N), 45.15 (CH2N), 51.24 (Cquat), 51.71 (=Cquat), 54.69 (=Cquat), 112.02 (CHarom), 114.43 (Br—Carom), 119.63 (CN), 120.15 (CN), 128.02 (CHarom), 131.90 (CHarom), 137.83 (Carom), 140.80 (Carom), 152.19 (=Cquat), 154.76 (=Cquat), 179.67 (O=C).6.Uiso(H) = 1.2Ueq(N), and C—H = 0.95–0.99 Å with Uiso(H) = 1.2 or 1.5Ueq(C). Both DMSO solvent molecules are disordered over two positions, with final occupancies of 0.90:0.10 for the first and 0.95:0.05 for the second molecule. In the first disordered DMSO molecule, the C17B and C18B atoms of the minor component were refined isotropically. The disordered atoms O2A/O2B, O3A/O3B, C19A/C19B and C20A/C20B were refined with anisotropic displacement parameters, constrained to be the same for both components. The S—C and S—O bond lengths in both disordered DMSO molecules were restrained to similarity.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022004741/zv2013sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022004741/zv2013Isup2.hklStructure factors: contains datablock(s) I. DOI: 2170241CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The correct data result is ‘IBL clear presentation time (23.59 ± 4.47 vs. 1026.80 ± 318.34 s) (p < 0.001)’ instead of ‘IBL clear presentation time (23.6 ± 4.4 vs. 23.6 ± 4.4 s) (p < 0.01)’. The results section should read as below:Results: An IBL was visible in 98% of patients in the ICGF‐based group, even with low doses of ICG. The ICGF‐based group was significantly associated with a shorter IBL clear presentation time (23.59 ± 4.47 vs. 1026.80 ± 318.34 s) (p < 0.001) and operative time (89.3 ± 31.6 vs. 112.9 ± 33.3 min) (p < 0.01) compared to the MID group. The incidence of postoperative prolonged air leaks was higher in the MID group than in the ICGF‐based group . There were no significant differences in bleeding volume, chest tube duration, postoperative hospital stays, surgical margin width, and other postoperative complications.The authors apologize for the error and any inconvenience it may have caused.


The title compound features a B—O—B bond angle of 132.75 (13)°. 40H36B2N4O5, the B—O—B bond angle is 132.75 (13) and the dihedral angle between the benzodiazborole rings is 73.02 (5)°. In the crystal, weak C—H⋯O interactions link the molecules.In the title compound, C Additionally, it is likely that a p-type electronic interaction exists between O1 and the adjacent boron atoms (B1A and B1B) that would serve to open up the bond angle substantially beyond the textbook angle of 109.5° for an O atom bearing two lone pairs of electrons. As a result of steric encumbrance, the B1A and B1B benzodiazaborole rings are angled away from one another to a near perpendicular orientation, with a plane-to-plane tilt of 73.02 (5)°. The dihedral angles between the B1A benzodiazaborole ring system and its pendant p-methoxybenzene rings are 80.49 (6) and 49.84 (7)° for the C7A and C14A rings, respectively. Comparable data for the B1B ring system and its pendant C7B and C14B rings are 78.32 (6) and 65.96 (7)°, respectively. The C atoms of the methoxy groups are all close to their respective ring planes: C13A [deviation = 0.333 (2) Å]; C20A [0.254 (2) Å]; C13B [−0.040 (2 Å)]; C20B [0.193 (2) Å].The B1In the crystal, weak C—H⋯O interactions Table 1 link theN1,N2-bis(4-methoxyphenyl)benzene-1,2-diamine , was obtained in 87% yield. The single-crystal X-ray structure of the diazaborole chloride has been deposited with the Cambridge Structural Database . After stirring overnight, a white precipitate of the triethylammonium chloride formed that was then filtered and discarded. The filtrate was dried under reduced pressure to give the crude product. The solid was extracted in a fritted glass filter with a minimum volume of benzene, and the filtrate was evaporated under reduced pressure to give the title compound in 85% yield.Under an anhydrous nitrogen atmosphere, a solution was prepared that contained 3.0 mmol of (II), four equivalents of triethylamine, and ∼200 ml of 1,2-dimethoxyethane. This solution was then treated with half an equivalent of water  global, I. DOI: 10.1107/S2414314620012481/hb4361Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620012481/hb4361Isup4.cmlSupporting information file. DOI: 2031384CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit is composed of half a binuclear silver complex located about a center of symmetry, a potassium cation and 2.6 disordered water molecules. The whole binuclear silver complex is generated by inversion symmetry with the pyrazine ring being located about an inversion centre. The ligand coordinates in a bis-tetradentate manner. The binuclear silver complex anions are linked via bridging Ag⋯S⋯Ag zigzag bonds, forming a network lying parallel to the bc plane. The networks are linked by Ocarboxylate⋯K+⋯Ocarboxylate bridging bonds to form a framework. The disordered water molecules are present near to the K+ cations.The reaction of AgNO In I this value is 2.550 (5) Å. For Ag—Ocarboxylate there were over 2,800 hits with the bond lengths varying from 1.967 to 3.089 Å . In I the Ag—Ocarboxylate bond lengths are almost equal; 2.470 (5) and 2.466 (6) Å. Finally for the Ag—S(CH2)2— bond-length type there were over 1,000 hits with the bond length varying from 2.361 to 3.583 Å . In I the Ag—S(CH2)2– bond lengths vary from 2.604 (2) to 2.926 (2) Å, both values involve the bridging atom S1, while distance Ag1—S2ii is 2.824 (2) Å  −x, −y + 1, −z + 1].The three chelate rings are far from flat, as indicated by the torsion angles given in Table 1K+⋯Ocarboxylate bonds lengths vary from 2.608 (6) to 2.751 (6) Å, and there is one weak contact K1⋯O1 at 3.289 (6) Å potassium] potassium]  to 2.8668 (13) Å in UBUPAK and from 2.8197 (14) to 3.0449 (15) Å in MUMPIW. In UBUPAK the K+ cation has a coordination number of 8 (KO8) and a distorted dodecahedral geometry, while in MUMPIW the K+ ion has a coordination number of 7 (KO6N) and has an edge-sharing pentagonal antiprism geometry. In I, the stronger K⋯O bond lengths are shorter and, owing to the presence of the disordered water molecules, it is not clear what the K+ ion coordination number or geometry are.Selected bond lengths and bond angles involving atom K1 are also given in Table 1 Å Fig. 3. A searcI, the networks of the binuclear silver complex anions are linked by the bridging Ocarboxylate⋯K+⋯Ocarboxylate bonds to form a framework ] tetrakis(sulfanediyl)}tetraacetato)}-bis[silver(I)]-bis[potassium] 5.2(hydrate)} (I):3  and 4L1H  were mixed in 20 ml of a 1M potassium acetate buffer solution. The mixture was left at 323 K under stirring and nitrogen conditions for 1 h. The mixture was then filtered and left to evaporate in air for six weeks, yielding yellow rod-like crystals of compound I (m.p. 553 K decomposition).AgNO16H16Ag2N2O8S4, K2, 5.2(H2O), Mw = 880.175 g mol−1: Calculated (%): C 21.88, H 2.99, N 3.18. Found (%): C 23.03, H 2.91, N 3.03. The small deviation is probably due to the loss of water molecules of crystallization.Analysis for CESI–MS: unstable under mass spectroscopy experimental conditions.−1) ν: 3401(s), 2938(m), 1599(s), 1385(s), 1223(m).IR . It was not possible to locate the H atoms of the disordered water molecules of crystallization. The residual electron density peaks of 1.14 and −1.10 eÅ3 are at distances of 0.96 and 0.91 Å, respectively, from atom Ag1.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314622000773/im4015sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2414314622000773/im4015Isup2.hklStructure factors: contains datablock(s) I. DOI: 2143798CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N‐hydroxyalkyl phenylbenzoisoquinolindiones (PBIQs) has been developed. Incubation of plant material of Xiphidium caeruleum with hydroxylamines of various chain lengths  resulted in 11 new 5‐hydroxy‐ and 5‐methoxy PBIQs with different N‐hydroxyalkyl side chain lengths. The antiproliferative effect and the cytotoxicity against HUVEC, K‐562, and HeLa cell lines of 26 previously reported PBIQs and the 11 newly synthesized N‐hydroxyalkyl PBIQs was determined for the first time. The results revealed that introducing long‐chain N‐aliphatic amine moieties improved the antiproliferative effect and cytotoxicity of PBIQs when compared to derivatives with N‐amino acids as side chains.A precursor‐directed approach to access  Direct incubation ofXiphidium caeruleumplant material with ω‐amino alcohols followed by extraction and purification results in N‐hydroxyalkyl phenylbenzoisoquinolindiones (PBIQs). Long‐chain derivatives show good antiproliferative effects together with low cytotoxicity. H‐benzo[de]isoquinoline‐1,3(2H)‐diones,Xiphidium caeruleumLachnanthes tinctoriaWachendorfia thyrsiflora.Isoquinolines, which represent one of the largest groups of naturally occurring alkaloids, show a wide spectrum of biological activities.X. caeruleum were derived from a series of enzymatic hydrolyses and spontaneous conversions from three native phenylbenzoisochromenone (PBIC) glucosides, 1–3, and plant proteinogenic amino acids  (see Supporting Information Table ST1).isoquinoline‐1,6‐dione (31): Orange powder. HPLC‐PDA‐HRESIMS gradient 2, tR 7.5 min; UV (MeCN‐H2O) λmax 206, 240, 320, 434 nm; 1H NMR data, see Supporting Information Table ST5; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 348.1237 [M+H]+ .2‐(4′′‐Hydroxybutyl)‐5‐hydroxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (32): Orange powder. HPLC‐PDA‐ESIMS gradient 2, tR 9.4 min; UV (MeCN‐H2O) λmax 206, 238, 322, 442 nm; 1H NMR data, see Supporting Information Table ST5; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 362.1394 [M+H]+ .2‐(4′′‐Hydroxybutyl)‐5‐methoxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (33): Orange powder. HPLC‐PDA‐HRESIMS gradient 2, tR 8.4 min; UV (MeCN‐H2O) λmax 196, 238, 320, 434 nm; 1H NMR data, see Supporting Information Table ST5; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 376.1541 [M+H]+ .2‐(6′′‐Hydroxyhexyl)‐5‐hydroxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (34): Orange powder. HPLC‐PDA‐HRESIMS gradient 2, tR 12.3 min; UV (MeCN‐H2O) λmax 224, 324, 444 nm; 1H NMR data, see Supporting Information Table ST5; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 390.1708 [M+H]+ .2‐(6′′‐Hydroxyhexyl)‐5‐methoxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (35): Orange powder. HPLC‐PDA‐HRESIMS gradient 2, tR 10.9 min; UV (MeCN‐H2O) λmax 206, 238, 320, 434 nm; 1H NMR data, see Supporting Information Table ST5; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 404.1863 [M+H]+ .2‐(8′′‐Hydroxyoctyl)‐5‐hydroxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (36): Orange powder. HPLC‐PDA‐HRESIMS gradient 2, tR 15.3 min; UV (MeCN‐H2O) λmax 206, 218, 238, 324, 444 nm; 1H NMR data, see Supporting Information Table ST5; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 418.2030 [M+H]+ .2‐(8′′‐Hydroxyoctyl)‐5‐methoxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (37): Orange powder. HPLC‐DAD‐ESIMS gradient 2, tR 14.1 min; UV (MeCN‐H2O) λmax 206, 238, 320, 434 nm; 1H NMR data, see Supporting Information Table ST6; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 432.2171 [M+H]+ .2‐(10′′‐Hydroxydecyl)‐5‐hydroxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (38): Orange powder. HPLC‐PDA‐HRESIMS gradient 2, tR 18.7 min; UV (MeCN‐H2O) λmax 208, 234, 324, 444 nm; 1H NMR data, see Supporting Information Table ST6; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 446.2326 [M+H]+ .2‐(10′′‐Hydroxydecyl)‐5‐methoxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (39): Orange powder. HPLC‐DAD‐ESIMS gradient 2, tR 17.6 min; UV (MeCN‐H2O) λmax 204, 238, 320, 434 nm; 1H NMR data, see Supporting Information Table ST6; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 460.2482 [M+H]+ .2‐(12′′‐Hydroxydodecyl)‐5‐hydroxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (40): Orange powder. HPLC‐DAD‐ESIMS gradient 2, tR 22.3 min; UV (MeCN‐H2O) λmax 206, 242, 324, 444 nm; 1H NMR data, see Supporting Information Table ST6; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 474.2649 [M+H]+ .2‐(12′′‐Hydroxydodecyl)‐5‐methoxy‐7‐phenyl‐2H‐benzo[de]isoquinoline‐1,6‐dione (41): Orange powder. HPLC‐DAD‐ESIMS gradient 2, tR 21.2 min; UV (MeCN‐H2O) λmax 204, 236, 320, 434 nm; 1H NMR data, see Supporting Information Table ST6; 13C NMR data, see Supporting Information Table ST7; HRESIMS m/z 488.2806 [M+H]+ .30–41 were dissolved in dimethylsulfoxide . Five replicates were investigated for each assay. Cell toxicity assays were conducted using a HeLa cell line (DSM ACC 57), and antiproliferative activity was assayed using HUVEC (ATCC CRL‐1730) and K‐562 cell lines (DSM ACC 10). The test substances were dissolved in DMSO before being diluted in the respective cell culture medium to concentrations of between 1 and 100 μg mL−1. The adherent cells were harvested in the logarithmic growth phase after soft trypsinization, using 0.25 % trypsin in phosphate‐buffered saline containing 0.02 % ethylenediaminetetraacetic acid. For each experiment, approximately 10,000 cells were seeded with 0.1 mL culture medium per well of a 96‐well microplate. HeLa cells were pre‐incubated for 48 h prior to the application of the test compounds to give subconfluent monolayers. Incubation was then conducted in a humidified atmosphere at 37 °C and 5 % CO2. In the case of K‐562 cells, the antiproliferative effect was determined using the CellTiter‐Blue1 assay.n HCl from the wells. The optical densities were measured at 660 nm in a SUNRISE microplate reader .To determine cell toxicity and antiproliferative activity, PBIQs PLUS  was performed as described earlier.Cell Death Detection ELISAAn overview of the compounds used for the bioassays together with their analytical data (MS and NMR) is provided in the Supporting Information. Furthermore, the results of the bioassays in graphical form and microscopic images of cells treated with PBIQs are given.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The chains are connected by C—F⋯π(ring), C=O⋯π(ring) and slipped π-stacking interactions. A Hirshfeld surface analysis of these interactions was performed.The asymmetric unit consists of two independent molecules differing in the orientation of the azido group. Each molecule forms N—H⋯O hydrogen-bonded chains extending along the  8H7FN4O, consists of two independent molecules differing in the orientation of the azido group. Each molecule forms N—H⋯O hydrogen-bonded chains along along the c-axis direction with its symmetry-related counterparts and the chains are connected by C—F⋯π(ring), C=O⋯π(ring) and slipped π-stacking interactions. A Hirshfeld surface analysis of these interactions was performed.The asymmetric unit of the title compound, C They have found valuable applications in medicinal chemistry  Å, C15⋯Cg1 = 3.902 (2) Å, C15=O2⋯Cg1 = 80.88 (13)°] as shown in Fig. 1p-tolyl analog  x, −y + z + A⋯O2i hydrogen bonds form parallel chains for the second independent molecule  −x + 1, −y + 1, −z + 1; (iii) −x + 2, −y + 1, −z + 2] interactions as well as by the C15=O2⋯Cg1 interaction noted above and weak, slipped π-stacking between centrosymmetrically related C1–C6 benzene rings  , H⋯H (13.5%), O⋯H/H⋯O (12.2%), C⋯H/H⋯C (11.9%) and F⋯H/H⋯F (9.7%), respectively. The two spikes in Fig. 6d primarily represent the N—H⋯O hydrogen bonds but their breadth at longer values of di + de than at the tips indicate the contributions from C—H⋯O hydrogen bonds. Fig. 7a showing all intermolecular interactions and Fig. 7b–7f those delineated into N⋯H/H⋯N (28.8%), H⋯H (18.2%), C⋯H/H⋯C (12.6%), F⋯H/H⋯F (12.6%) and O⋯H/H⋯O (11.6%), respectively. Although the ordering of interactions based on their percentage of the total is not the same as in the other molecule, the percentages are not greatly different between the two and the corresponding plots are very similar type by type.A Hirshfeld surface analysis was performed with 6.N-(4-fluorophenyl)acetamide (0.011 mol), and sodium azide (0.015 mol) were dissolved in a mixture of ethanol/water (70:30) and refluxed for 24 h at 353 K. After completion of the reaction , the 2-azido-N-(4-fluorophenyl)acetamide that precipitated was filtered off and washed with cold water. A portion of the product was dissolved in hot ethanol, the solution was filtered, and the filtrate was left undisturbed for 7 days to form colorless, thick plate-like crystals.2-Chloro-−1) 3254 υ (N—H amide), 1027 υ (N—C amide), 1660 υ (C=O amide), 3073 υ(C—Harom), 1175 υ(C—N), 2961 υ, 2109 υ (N3), 1H NMR (DMSO–d6) δ ppm: 4.02 , 6.93–7.11 , 10.05 , 13C NMR (DMSO–d6) δ ppm: 51.18 (CH2), 131.47 (Carom—N), 113.90–120.86 (Carom); 165.71 (C=O); HRMS (ESI–MS) (m/z) calculated for C8H7FN4O 194.18; found 194.1165.Yield 69%, m.p. 358–360K, FT–IR  global, I. DOI: 10.1107/S2056989022006764/vm2266Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022006764/vm2266Isup3.cmlSupporting information file. DOI: 2183049CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The ability of 4-[(benzylamino)carbonyl]-1-methylpyridinium bromide salt to form hydrates was studied. Hirshfeld surfaces analysis was performed for identification of intermolecular interactions. 14H15N2O+·Br−·0.5H2O, was studied by single-crystal and powder X-ray diffraction methods. In the asymmetric unit, two organic cations of similar conformation, two bromide anions and one water molecule are present. In the crystal, N—H⋯Br hydrogen bonds link the cations and anions. The formation of a set of intermolecular C—H⋯Br and C—H⋯π interactions result in double chains extending parallel to [011]. A Hirshfeld surface analysis showed high contributions of H⋯H and C⋯H/H⋯C short contacts to the total Hirshfeld surfaces of the cations.The hemihydrate of 4-[(benzylamino)carbonyl]-1-methylpyridinium bromide, C Polymorphic screening for this salt resulted in the crystallization of a hemihydrate. In this communication we present the molecular and crystal structures of 4-[(benzylamino)carbonyl]-1-methylpyridinium bromide hemihydrate, (C2.A and B), two bromide anions (A and B) and one water molecule . The contribution of C⋯H/H⋯C short contacts is also significant . The Br⋯H/H⋯Br and O⋯H/H⋯O interactions contribute to the total Hirshfeld surface in the same way .Intermolecular interactions were analysed using Hirshfeld surface analysis and two-dimensional fingerprint plots by using rm Fig. 4. The redrm Fig. 4. The mairm Fig. 4g,h. Thent Fig. 5i,j. The5.et al., 2016et al., 2017sp3 and Csp3—Caryl bonds.A search of the Cambridge Structural Database . A Rietveld refinement carbonyl]-1-methylpyridinium iodide , silver bromide  and 700 ml of water were loaded into a glass flask. The mixture was stirred for 72 h, and the resulting precipitate was filtered off. The solvent was evaporated under reduced pressure. To the precipitate were added 300 ml of acetonitrile and refluxed for 2 h. The reaction then was spontaneously cooled to a temperature of 303 K and the precipitate filtered off and rinsed on the filter with 50 ml of cooled acetonitrile. The product was dried at 313 K for 12 h. Yield: 14 g of 4-[(benzylamino)carbonyl]-1-methylpyridinium bromide (28%); colourless crystals.8.Uiso(H) = 1.5Ueq for methyl groups and with Car—H = 0.93 Å, Csp2—H = 0.97 Å, Uiso(H) = 1.2Ueq for all other hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989022003784/wm5623sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022003784/wm5623Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022003784/wm5623Isup3.cmlSupporting information file. DOI: 2164796CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The ‘tromancenium-8-carboxylic acid’ entity with a hexafluoridophosphate counter-ion represents a rare case of a cationic carboxylic acid. 7H7)(C6H5O2)]PF6 or [(Cht)Mn(Cp’CO2H)]PF6, with Cht = cycloheptatrienyl and Cp’ = C5H4, is an air-stable, purple, heteroleptic, cationic sandwich complex with manganese in oxidation state +I and π-coordinating cycloheptatrienyl and cyclopentadienyl ligands. The latter ligand carries the carboxylic acid functionality. This ‘tromancenium-8-carboxylic acid’ with hexafluoridophosphate as counter-ion represents a rare case of a cationic carboxylic acid. Structurally, this organometallic carboxylic acid displays the common motif of planar Osp2⋯H—Osp3/Osp3—H⋯Osp2 hydrogen-bonded carboxylic acid dimers with anti-oriented metallocenyl moieties, the cationic charge of which is balanced by octahedrally shaped hexafluoridophosphate anions. Positional disorder is observed in the cycloheptatrienyl ring and the PF6− anion.The title compound, [Mn(C As a result of the instability against nucleophiles of the parent compound tromancenium hexafluoridophosphate  Å is typical for a carbon–carbon single bond. The C13—O1 bond length of 1.205 (10) Å is shorter than the C13—O2 bond length of 1.303 (10) Å, which is coherent with the expectations.The molecular entities of the title compound are shown in Fig. 1y Table 1, with tret al., 2014Cht/Cp bond lengths in the title compound. The C=O bond in cobaltocenium carboxylic acid is of the same length as the C—O bond, due to disorder.The comparable organometallic compound cobaltocenium carboxylic acid hexafluoridophosphate (Vanicek 6−), which fill the space within the packing of the dimers  were added. The solvents were removed on a rotary evaporator and the crude material dried in vacuo. The product was dissolved in acetonitrile and filtered through a folded paper filter. Acetonitrile was removed on a rotary evaporator and the product was dried in vacuo giving pure 8-tromancenium carboxylic acid hexafluoridophosphate in 92% yield . Single crystals were obtained by diffusion crystallization in acetonitrile out of diethyl ether at room temperature.A round-bottom flask was charged with 0.0563 g of 8-carbomethoxytromancenium hexafluoridophosphate  δ = 4.89 , 5.21 , 6.93 ; signal of CO2H not observed due to rapid exchange. 13C NMR  δ = 78.6 (ipso-carbon of Cp), 79.4 (C10/C11 of Cp), 80.3 (C9/C12 of Cp), 99.0 (C1–7 of Cht), 156.4 (CO2H). 55Mn NMR  δ = 529. IR : 3000 (νO—H + νC—H), 1696 (νC=O), 1489, 1448, 1413, 1375 (νC—OH + νC=C), 815 (νP—F), 749 ), 600, 554 , 467, 437 (νMn). HRMS  255.0211 ([M − PF6]+), calculated for C13H12O2Mn: 255.0212. UV–vis  λmax1 = 283, λmax2 = 559. Cyclic voltammetry (CV): ΔE1/2 (Mn+/Mn2+) = 1.00 V versus ferrocene/ferrocenium+ (irreversible).Properties: m.p.: 395.8 K dec. d = 0.83 Å). A positional disorder in a ratio of 1:1 for the carbon atoms and attached hydrogen atoms of the seven-membered ring: C1–C7: C1A–C7A was considered; the corresponding carbon atoms were refined with isotropic displacement parameters. A further positional disorder of all fluorine atoms of the PF6− anion was refined in ratio 45:55 for F1–F6:F1A—F6A with anisotropic displacement parameters. In an alternative model, the crystal structure was also refined in the non-centrosymmetric space group P1 with a new data set, for which TWINABS  in the P1 model and several remaining electron-density peaks between the carbon atoms of the two seven-membered rings clearly show that the disorder will be retained in the non-centrosymmetric space group. Hence, the latter was discarded and the centrosymmetric model used for final processing.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623001074/wm4180sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314623001074/wm4180Isup2.hklStructure factors: contains datablock(s) I. DOI: 2239883CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Correction: “Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike‐in standard” by Olivier Zemb | Caroline S. Achard | Jerome Hamelin | Marie‐Léa De Almeida | Béatrice Gabinaud | Laurent Cauquil | Lisanne M.G. Verschuren | Jean‐Jacques Godon published online on 11 January Figure 1 of the original version of published article has been replaced. The correct figure is placed below: In the original version, in the text (section 2.3) “forward and reverse primers E 5′‐CAGATGTGAAATCATCGATCG/5′‐CCGATTTCAATCGTACACCTG” should read “forward and reverse primers E 5′‐ACGTAGAATTCCAGGTGTACGAT/5′‐ACCTGAGCGTCAGTCTTCGT”.


Scientific Reports 10.1038/s41598-021-82599-1, published online 04 February 2021Correction to: The original version of this Article contained an error in the Methods section, where “7.8 × 300 mm” was incorrectly given as “4.6 × 300 mm”.In the Methods section, under the subheading ‘Size-exclusion chromatography’,“A SEPAX SRT SEC-1000 column (4.6 × 300 mm) and guard column  were used for all SEC-MALS experiments.”now reads:“A SEPAX SRT SEC-1000 column (7.8 × 300 mm) and guard column  were used for all SEC-MALS experiments.”The original Article has been corrected.


The a new polymorph of the title compound is reported in which the C—H⋯O hydrogen bonds and π-π stacking interactions link molecules into the layers in the crystal. 14H8Br3N3O2, (form-2) was obtained in the same manner as the previously reported form-1 . The structure of the new polymorph is stabilized by a C—H⋯O hydrogen bond that links molecules into chains. These chains are linked by face-to-face π–π stacking interactions, resulting in a layered structure. Short inter-molecular Br⋯O contacts and van der Waals interactions between the layers aid in the cohesion of the crystal packing. In the previously reported form-1, C—H⋯Br interactions connect molecules into zigzag chains, which are linked by C—Br⋯π interactions into layers, whereas the van der Waals interactions between the layers stabilize the crystal packing of form-2. Hirshfeld molecular surface analysis was used to compare the intermolecular interactions of the polymorphs.A new polymorph of the title compound, C al. 2022. Acta Cr Crystal Explorer 17.5  to +1.1715 (blue) a.u. , H⋯H (12.8%), C⋯H/H⋯C (11.5%) and O⋯H/H⋯O (10.6%) contacts are shown in Fig. 6b–e, while the numerical details for the shortest contacts are given in Table 2viz. Br⋯H/H⋯Br, H⋯H, C⋯H/H⋯C and O⋯H/H⋯O contacts -1--2-(4-bromophenyl)diazene unit showed that the ten closest are those of CSD refcodes HEHKEO (I)          , resulting in zigzag C(8) chains along the [100] direction. These chains are connected by C—Br⋯π interactions into layers parallel to (001). van der Waals interactions between the layers contribute to the crystal cohesion.C—H⋯Br interactions connect the molecules in the crystal of the form-1 polymorph of the title compound, (II) are joined into layers parallel to (011) by C—H⋯O and C—H⋯F hydrogen bonds. C—Br⋯π and C—F⋯π contacts, as well as π–π stacking interactions, strengthen the crystal packingThe molecules in (III) are connected into chains running parallel to [001] by C—H⋯O hydrogen bonds. C—F⋯π contacts and π–π stacking interactions help to consolidate the crystal packing, and short Br⋯O [2.9828 (13) Å] distances are also observed.The molecules in the crystal of (IV), the molecules are linked into inversion dimers via short halogen–halogen contacts . No other directional contacts could be identified, and the shortest aromatic ring-centroid separation is greater than 5.25 Å.In the crystal of (V) and (VI), the molecules are linked through weak X⋯Cl contacts [X = Cl for (V) and Br for (VI)], C—H⋯Cl and C—Cl⋯π interactions into sheets lying parallel to (001).In the crystals of (VII), the molecules are stacked in columns along [100] via weak C—H⋯Cl hydrogen bonds and face-to-face π–π stacking interactions. The crystal packing is further consolidated by short Cl⋯Cl contacts.In the crystal of (VIII), molecules are linked by C—H⋯O hydrogen bonds into zigzag chains running parallel to [001]. The crystal packing also features C—Cl⋯π, C—F⋯π and N—O⋯π interactions.In (IX), C—H⋯N and short Cl⋯Cl contacts are observed, and in (X), C—H⋯N and C—H⋯O hydrogen bonds and short Cl⋯O contacts occur.In -1-(4-bromophenyl)-2-(3-nitrobenzylidene)hydrazine (1 mmol), tetramethylethylenediamine , CuCl  and CBr4 (4.5 mmol). After 1–3 h , the reaction mixture was poured into a 0.01 M solution of HCl , and extracted with dichloromethane (3 × 20 mL). The combined organic phase was washed with water (3 × 50 mL), brine (30 mL), dried over anhydrous Na2SO4 and concentrated in vacuo using a rotary evaporator. The residue was purified by column chromatography on silica gel using appropriate mixtures of hexane and dichloromethane (3/1–1/1). Crystals suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution. Red solid (62%); m.p. 391 K. Analysis calculated for C14H8Br3N3O2 (M = 489.95): C 34.32, H 1.65, N 8.58; found: C 34.29, H 1.66, N 8.55%. 1H NMR  δ 7.90–7.44 . 13C NMR  δ 150.88, 148.57, 148.12, 132.81, 132.47, 132.25, 130.04, 126.40, 125.30, 124.53, 123.57, 94.10. ESI-MS: m/z: 490.91 [M + H]+.This dye was synthesized according to the reported method (Akkurt 6.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022007113/yk2172sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022007113/yk2172Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022007113/yk2172Isup3.cmlSupporting information file. DOI: 2189348CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: