In the title compound, intermolecular N—H⋯O hydrogen bonds link the molecules into a three-dimensional supramolecular network. H-pyridazin-3-one derivative. The molecule, C18H16N2O, is not planar; the benzene and pyridazine rings are twisted with respect to each other, making a dihedral angle of 11.47 (2)°, and the toluene ring is nearly perpendicular to the pyridazine ring, with a dihedral angle of 89.624 (1)°. The molecular conformation is stabilized by weak intramolecular C—H⋯N contacts. In the crystal, pairs of N—H⋯O hydrogen bonds link the molecules into inversion dimers with an R22(8) ring motif. The intermolecular interactions were investigated using Hirshfeld surface analysis and two-dimensional (2D) fingerprint plots, revealing that the most important contributions for the crystal packing are from H⋯H (56.6%), H⋯C/C⋯H (22.6%), O⋯H/H⋯O (10.0%) and N⋯C/C⋯N (3.5%) interactions.In this paper, we describe the synthesis of a new dihydro-2 Pyridazinone is an important pharmacophore possessing a wide range of biological applications . Weak C—H⋯O hydrogen bonds and weak off-set π-stacking stabilize the packing. In the crystal, hydrogen bonds link the chains into a two-dimensional (2D) network parallel to (011) ], with a centroid-to-centroid distance of 3.8333 (18) Å and a slippage of 1.460 Å (Cg1 is the centroid of the C9–C11/N1/N2 ring and Cg3 is the centroid of the C13–C18 ring) .The molecules are connected two-by-two through N1—H1⋯O1 hydrogen bonds Table 2, with a g) Fig. 2a.et al., 2016H)-one (see A in Schemep-tolylpyridazin-3(2H)-one  to 1.4188 a.u. (blue). The 3D dnorm surface of the title complex is illustrated in Figs. 3a) and 4dnorm values on the surface correspond to the N—H⋯O interactions  is due to short interatomic H⋯H contacts. In the absence of C—H⋯π interactions in the crystal, the pair of characteristic wings in the fingerprint plot representing H⋯C/C⋯H contacts (22.6% contribution to the HS) have a symmetrical distribution of points , with the tips at de + di = 2.797 Å. The O⋯H  contacts contribute 10% to the HS and have a symmetrical distribution of points, with the tips at de + di = 1.853 Å. The contribution of the other contact to the Hirshfeld surface is N⋯C/C⋯N (3.5%). The Hirshfeld surface representations with the function dnorm plotted on the surface are shown for the H⋯H, H⋯C/C⋯H, H⋯O/O⋯H, C⋯C and H⋯N/N⋯H interactions in Figs. 6et al., 2015The Hirshfeld surface analysis . The convex blue regions on the shape-index symbolize hydrogen-donor groups and the concave red regions symbolize hydrogen-acceptor groups. The π–π interactions on the shape-index map of the Hirshfeld surface are generally indicated by adjacent red and blue triangles.A shape-index map of the title compound was generated in the range −1 to 1 Å Fig. 3b. The cc). This shows large regions of green indicating a relatively flat surface area (planar), while the blue regions indicate areas of curvature. The presence of π–π stacking interactions is also evident in the flat regions around the rings on the Hirshfeld surface plotted over curvedness (see the Supramolecular features section above).A curvedness map of the title compound was generated in the range −4 to 0.4 Å Fig. 3c. This H)-one and  of 4-methylbenzaldehyde in ethanol (30 ml), sodium hydroxide  was added. The solvent was evaporated under vacuum and the residue was purified through silica-gel column chromatography using hexane/ethyl acetate (7:3 v/v). Slow evaporation at room temperature leads to single crystals.To a solution  of 6-phenyl-4,5-dihydropyridazin-3(2Uiso(H) = 1.5Ueq(C) for methyl, C—H = 0.96 Å and Uiso(H) = 1.2Ueq(C) for methylene, C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C) for aromatic and C—H = 0.98 Å and Uiso(H) = 1.2Ueq(C) for methine H atoms. Crystal data, data collection and structure refinement details are summarized in Table 3H atoms were fixed geometrically and treated as riding, with C—H = 0.97 Å and 10.1107/S2056989019011551/mw2146sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019011551/mw2146Isup3.hklStructure factors: contains datablock(s) I. DOI: 1947718CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-indene-1,3(2H)-dione (ID[1]) and (E)-2-{3-[4-(dimethylamino)phenyl]allylidene}-1H-indene-1,3(2H)-dione (ID[2]), with donor–π-bridge–acceptor structures, are almost planar for the molecule with a short π-bridge (ID[1]) but less planar for the molecule with a longer bridge (ID[2]).The title pull–push chromophores, 2-[4-(dimethylamino)benzylidene]-1 H-indene-1,3(2H)-dione, C18H15NO2 (ID[1]) and (E)-2-{3-[4-(dimethylamino)phenyl]allylidene}-1H-indene-1,3(2H)-dione, C20H17NO2 (ID[2]), have donor–π-bridge–acceptor structures. The molecule with the short π-bridge, ID[1], is almost planar while for the molecule with a longer bridge, ID[2], is less planar. The benzene ring is inclined to the mean plane of the 2,3-dihydro-1H-indene unit by 3.19 (4)° in ID[1] and 13.06 (8)° in ID[2]. The structures of three polymorphs of compound ID[1] have been reported: the α-polymorph  is non-centrosymmetric (space group P21), which suggests potential NLO properties for this crystalline material. In both compounds, there is short intramolecular C—H⋯O contact present, enclosing an S(7) ring motif. In the crystal of ID[1], molecules are linked by C—H⋯O hydrogen bonds and C—H⋯π interactions, forming layers parallel to the bc plane. In the crystal of ID[2], molecules are liked by C—H⋯O hydrogen bonds to form 21 helices propagating along the b-axis direction. The molecules in the helix are linked by offset π–π interactions with, for example, a centroid–centroid distance of 3.9664 (13) Å (= b axis) separating the indene rings, and an offset of 1.869 Å. Spectroscopic and electrochemical measurements show the ability of these compounds to easily transfer electrons through the π-conjugated chain.The title pull–push chromophores, 2-[4-(dimethylamino)benzylidene]-1kova 1978. Kristalkova 1980. Kristalkaya 1980. Kristal Applications of pull–push chromophores include non-linear optics  are important in many areas of materials chemistry, especially organic electronics and optoelectronics. Applications of pull–push molecules can be related to their properties such as intramolecular charge transfer and specific molecular arrangements in the solid state. Intramolecular charge transfer from donor to acceptor The molecular structures of ID[1] and ID[2] are illustrated in Fig. 1et al., 2019Both molecules have acceptor–π-bridge–donor structures. It was found, as in our previous studies  being inclined to the mean plane of the indene ring system (C1–C9) by 3.19 (4)°. In ID[2] the deviation from planarity is somewhat larger with the benzene ring (C10–C15) being inclined to the mean plane of the indene ring system (C1–C9) by 13.06 (8)°; see further details in Table 1Molecules of ID[1] and ID[2] have significant dipole moments, which is very common for NLO chromophores. Because of this, molecules have a trend to antiparallel packing, which is observed in the crystal structures of both ID[1] and ID[2].ca 84.47° between them. The molecules, which stack in an antiparallel or head-to-tail fashion, are linked by C—H⋯O hydrogen bonds  Å  separating the indene ring systems (C1–C9), with an offset of 1.869 Å.In the crystal of ID[2], molecules form stacks with parallel molecular positions, and shifted positions of stacks extended along the on Fig. 3. Here, ts Table 3, forminget al., 2019Absorbance spectra were obtained for both ID[1] and ID[2] in chloroform and acetonitrile. For donor–acceptor polyenes, the dominating feature of the absorbance spectrum is the π–π* transition that results from charge transfer from donor to acceptor. According to recent studies (Bogdanov a), however, when swept to −1.9 V the reduction is only partly reversible . This represents the ability of the compound to ‘easily’ transfer electrons through the chain from donor towards acceptor.Donor–acceptor polyenes can be characterized by electrochemical measurements to show their ability to transfer electrons. The voltammagrams Fig. 5 demonstrle Fig. 5b. This 2+/0  in dichloromethane with 0.1 nM Bu4NPF6). Measurements were recorded at 50 mV s−1 using a BAS Potentiostat using a glassy carbon working electrode, Pt wire auxilliary electrode and a Ag/AgCl reference electrode.Note: cyclic voltammagrams of ID[1] were made against FeCpet al., 2016p-diethylaminobenzylidene)-1,3-indandione -dione -dione , 1.66 (6)/1.49 (9) and 5.71 (9)°, for TELWEN, QENYEQ/QENYEQ01 and BIQYUY, respectively, compared to 3.19 (4)° in ID[1].A search of the Cambridge Structural Database , while in the γ polymorph it is acentric (space group Pna21). The crystal structure of ID[1] we obtained corresponds to the β polymorph, i.e. the centrosymmetric modification MBYINO02. The dihedral angles between the benzene and indene rings for two independent molecules in MBYINO are ca 4.35 and 7.79°, compared to ca 7.36° in MBYINO01, and 3.54° in MBYINO02 (cf. 3.19 (4)° in the present crystal structure analysis of ID[1]).Also, out of all 27 hits there are three hits, -dione ° in ID[2]. In ZIGPIR this dihedral angle is smaller at 8.6 (3)°.A search of the CSD for the substructure of ID[2] yielded nine hits. Only two structures are similar to that of ID[2]. The first, 2-{3-[4-(dimethylamino)phenyl]prop-2-yn-1-ylidene}-1et al., 2019For the synthesis of the title compounds, two aldehydes were used: 4-(dimethylamino)benzaldehyde  and 4-(dimethylamino)cinnamaldehyde (A2), which was synthesized as described previously benzylidene]indane-1,3-dione (ID[1]): Aldehyde A1  and 1,3-indanedione  were suspended in 100 ml of absolute ethanol. The mixture was gently heated until the solids had dissolved. After about 10 min of stirring the dissolution was complete, and a red crystalline precipitate began forming on the walls of the flask. The reaction mixture was stirred vigorously overnight, and the resulting product was collected by filtration then washed with cold ethanol and hexanes to give shiny dark-red crystals . ID[1] can be purified by recrystallization using numerous solvent systems , many of which afforded single crystals. 1H NMR  δ 8.52 , 7.91–9.73 , 7.71 , 6.77 , 3.14  ppm. 13C NMR  δ 191.6, 190.1, 154.5, 147.3, 142.7, 140.3, 138.2, 134.8, 134.5, 123.3, 122.7, 122.6, 122.2, 111.7, 40.3 ppm.Synthesis of (E)-2-{3-[4-(dimethylamino)phenyl]allylidene}indane-1,3-dione (ID[2]): Aldehyde A2 , 1,3-indanedione  and piperidine  in ethanol (50 ml) were mixed and treated as for the synthesis of ID[1]. The crude product obtained was collected by filtration and washed with cold ethanol before being recrystallized from ethanol to give incredibly shiny and thin purple actinic crystals . They were washed with hexane and dried under vacuum. 1H NMR  δ 8.25 , 7.90–7.86 , 7.76–7.73 , 7.60 , 7.60 , 7.34 , 6.72 , 3.08  ppm.Uiso(H) = 1.5Ueq(C-methyl) and 1.2Uiso(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S205698901901329X/su5515sup1.cifCrystal structure: contains datablock(s) ID1, ID2, Gobal. DOI: 10.1107/S205698901901329X/su5515ID1sup2.hklStructure factors: contains datablock(s) ID1. DOI: 10.1107/S205698901901329X/su5515ID2sup3.hklStructure factors: contains datablock(s) ID2. DOI: Click here for additional data file.10.1107/S205698901901329X/su5515ID1sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901901329X/su5515ID2sup5.cmlSupporting information file. DOI: 1956419, 1956418, 1956418, 1956419CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N‐Methylation of lysyl residues is widely observed on histone proteins. Using isolated enzymes, we report mechanistic and structural studies on histone lysine demethylase (KDM)‐catalysed demethylation of Nε‐methylated lysine 26 on histone 1 isotype 4 (H1.4). The results reveal that methylated H1.4K26 is a substrate for all members of the KDM4 subfamily and that KDM4A‐catalysed demethylation of H1.4K26me3 peptide is similarly efficient to that of H3K9me3. Crystallographic studies of an H1.4K26me3:KDM4A complex reveal a conserved binding geometry to that of H3K9me3. In the light of the high activity of the KDM4s on this mark, our results suggest JmjC KDM‐catalysed demethylation of H1.4K26 may be as prevalent as demethylation on the H3 tail and warrants further investigation in cells. Our findings reveal that demethylation of methylated lysyl residues in H1.4K26 peptide fragments is catalysed by all members of the human KDM4 subfamily of JmjC KDMs (KDM4A‐E), supporting previously reported cell‐based studies (predominantly with mouse enzymes) Escherichia coli . Sequences are given in Table Peptides were produced as C‐terminal amides using a Liberty Blue automated microwave peptide synthesiser , using standard fluorenylmethyloxycarbonyl‐mediated solid‐phase chemistry and NovaPEG rink amide resin . Following synthesis, peptides were cleaved from the resin by incubation with trifluoroacetic acid/water/triisopropylsilane/dimethoxybenzene (92.5 : 2.5 : 2.5 : 2.5) for 3 h followed by precipitation with ice‐cold diethyl ether. For kinetic experiments, lyophilised peptides were purified by reverse‐phase high‐performance liquid chromatography using a Vydac C18 column (Solvent A: 0.1% trifluoroacetic acid in Hm) were incubated with peptides  ammonium sulfate (10 μm), sodium ascorbate (100 μm) and 2‐oxoglutaric acid (200 μm). Reactions with KDM3A and KDM7A also contained 1 mm tris(2‐carboxyethyl)phosphine hydrochloride. No enzyme reactions were included as negative controls. Reactions were incubated for 1 h at 37 °C before being quenched with 1 : 1 (v/v) methanol. Analysis was carried out by MALDI‐TOF MS , with demethylation observed as mass shifts of 14 Da.Recombinant proteins (1 μ1H observation and were carried out as previously described m), 2OG (400 μm), H1.4(18–32)K26me3 peptide (400 μm), Fe(II) ammonium sulfate (100 μm) and ascorbate (1 mm) in 50 mm ammonium formate, pH 7.5, 500 mm NaCl in 10% v/v D2O) were prepared in a microcentrifuge tube , before being transferred to an NMR tube. The samples were then analysed over time using automated routines; each time‐point corresponded to one experiment accumulating 16 transients . The solvent resonance was depleted by excitation sculpting using a 180° sinc pulse (duration = 2 ms) The NMR analyses employed a Bruker Avance III 700 MHz spectrometer with an inverse TCI cryoprobe optimised for +‐coupled) assay for quantification of the formaldehyde reaction byproduct, as previously described m HEPES, pH 7.5, 0.01% Tween‐20, with addition of Fe(II) ammonium sulfate (10 μm), sodium ascorbate (100 μm), 2‐oxoglutarate (200 μm), NAD+ (500 μm), KDM4 enzymes  and FDH enzyme  in a 30‐μL volume in black 384‐well plates. Specific activities were measured using a 100‐μm peptide. For Michaelis–Menten experiments, peptide concentrations were varied. Reactions were monitored using a PHERAstar FS plate reader  with 355 nm excitation and 460 nm emission. Kinetic parameters were calculated from the reaction rate during the initial linear phase of formaldehyde production, which were used to calculate specific activities or fitted to Michaelis–Menten equations using graphpad prism .Kinetic parameters for KDM4 enzymes were determined by use of an FDH /NAD1–359.Ni(II).H1.4(18–32)K26me3.NOG were obtained in sitting drops grown at 4 °C with a ratio of 1 : 2 sample to well solution [0.1 m MIB buffer ] −1 KDM4A, 5 mm H1.4(18–32)K26me3, 5 mm NOG and 4 mm NiCl2. Crystals were cryoprotected with 25% glycerol then flash‐frozen in liquid N2. Data were collected using a single crystal at 100 K at the Diamond I04‐1 MX beam line and were processed with HKL2000 http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2OX0) and was refined by alternative cycles of CNS http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6H8P.Co‐crystals of KDM4A).H1.418–K26me3.NO2N‐TPVKKKARKSAGAAK‐CONH2), but containing either a Nε‐tri‐, di‐ or monomethyllysine at K26 were synthesised  and fragment peptides Table  37. Thresed Fig. . Activitsed Fig. . After iM4D Fig.  15, 43. M4D Fig. , low levM4D Fig. .Given the activity observed with KDM4E and KDM7A, we decided to investigate the intra‐subfamily conservation of this demethylation activity. The demethylation activity of the other subfamily members (KDM4A‐D and KDM7B) was therefore screened. For all KDM4 subfamily members, we observed H1.4K26 demethylation activity similar to that already observed with KDM4E Table ; howeverNε‐Trimethylated H3K4 binds to the plant homeobox domain of KDM7B, targeting the Nε‐dimethyllysine at H3K9 to the catalytic domain, and thus promoting demethylation di‐Nε‐methyllysine at K26 and tri‐Nε‐methyllysine at K21 K21me3K26me2) and the H1.4 peptides methylated only at K26 .K26 Fig. , but no 1H NMR time course analyses with KDM4A and the trimethylated H1.4K26me3 peptide confirmed time‐dependent demethylation of the Kme3 residue  were determined for comparison ‐coupled demethylation assay, which monitors the formation of NADH during FDH‐catalysed oxidation of formaldehyde son Figs D, S14. T+ and FDH were analysed over time and kinetic parameters were determined by recording the initial reaction rates of NADH production  at varying peptide concentration. Similar experiments were carried out with H3K9me3 and H3K36me3 substrate peptides and the obtained values compared (Table KM (32.3 ± 6.4 μm and 25.5 ± 4.9 μm respectively) and kcat (0.32 ± 0.049 s−1 and 0.31 ± 0.045 s−1 respectively) values. Demethylation of the H3K36me3 peptide was the least efficient, with higher KM (66.8 ± 5.4 μm) and lower kcat (0.12 ± 0.004 s−1) values than with the other two peptides.Michaelis–Menten kinetics were then conducted with KDM4A. Samples containing KDM4A, 2OG, ascorbate, H1.4K26me3 peptide, NADN‐oxalylglycine:H1.4K26me3 peptide complex was solved to a resolution of 1.98 Å (Note: Ni(II) was used for crystallisation in place of the catalytically relevant Fe(II) due to its oxidative stability; N‐oxalylglycine (NOG) is a 2OG mimetic inhibitor) Crystallographic analyses were undertaken to investigate the binding mode of trimethylated H1.4K26 in the KDM4A active site. An X‐ray crystal structure of a KDM4A:Ni(II):http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2OQ6http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2P5B‐ to C‐ directionality through the active site. The two residues N‐terminal to the trimethyllysine residue  adopt a similar conformation/orientation in all three structures, with backbone hydrogen bonds between KDM4A E169 and the peptide residues at the −1 and −2 positions  in its sequence. Thus, in the K36 peptide, the backbone carbonyl of the H3K36 to K37 amide bond is ‘flipped’ ~180° compared to the analogous amide in the other two substrates :NOG:H3K9me3 peptide complex  than either H3K9me3 or H1.4K26me3, binds differently at the KDM4A active site N‐methylated substrate lysine for H3K9/H1.4K26 versus H3K36 K26me3 crystal structure.Fig. S2. KDM4E catalyses lysine demethylation at H1.4K26.Fig. S3. KDM2A does not catalyse lysine demethylation at H1.4K26 under the tested conditions.Fig. S4. KDM3A does not catalyse lysine demethylation at H1.4K26 under the tested conditions.Fig. S5. KDM5C does not catalyse lysine demethylation at H1.4K26 under the tested conditions.Fig. S6. KDM6B does not catalyse lysine demethylation at H1.4K26 under the tested conditions.Fig. S7. KDM7A catalyses lysine demethylation at H1.4K26.Fig. S8. KDM4A catalyses lysine demethylation at H1.4K26.Fig. S9. KDM4B catalyses lysine demethylation at H1.4K26.Fig. S10. KDM4C catalyses lysine demethylation at H1.4K26.Fig. S11. KDM4D catalyses lysine demethylation at H1.4K26.Fig. S12. PHF8/KDM7B only catalyses lysine demethylation at H1.4K26 at high concentration.Fig. S13. Analysis of KDM4A demethylation by 1H NMR.Fig. S14. Specific activity determination for KDM4 enzymes.Table S1. Peptide sequences used in this study.Table S2. Crystallographic data processing and refinement statistics.Click here for additional data file.


Brmind⋯ODio, C—HBrmind⋯OEthy, C—HChlethy⋯ODio and C—HChlethy⋯OChlethy  hydrogen bonds link the molecules into a three-dimensional structure, enclosing The title compound consists of the 5-bromoindoline-2,3-dione unit linked by a 1-{2-[2-(2 chloroethoxy)ethoxy]ethyl} moiety. In the crystal, intermolecular C—H 14H15BrClNO4, consists of a 5-bromoindoline-2,3-dione unit linked to a 1-{2-[2-(2-chloroethoxy)ethoxy]ethyl} moiety. In the crystal, a series of C—H⋯O hydrogen bonds link the molecules to form a supramolecular three-dimensional structure, enclosing R22(8), R22(12), R22(18) and R22(22) ring motifs. π–π contacts between the five-membered dione rings may further stabilize the structure, with a centroid–centroid distance of 3.899 (2) Å. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (28.1%), H⋯O/O⋯H (23.5%), H⋯Br/Br⋯H (13.8%), H⋯Cl/Cl⋯H (13.0%) and H⋯C/C⋯H (10.2%) interactions. Hydrogen bonding and van der Waals interactions are the dominant interactions in the crystal packing. Density functional theory (DFT) optimized structures at the B3LYP/6-311G level are compared with the experimentally determined molecular structure in the solid state. The HOMO—LUMO behaviour was elucidated to determine the energy gap. The chloroethoxyethoxyethyl side chain atoms are disordered over two sets of sites with an occupancy ratio of 0.665 (8):0.335 (6).The title compound, C They are very interesting chemical compounds because of their potential applications in different fields. The most common heterocycles contain nitrogen and oxygen  and B (N1/C1/C6–C8), are oriented at a dihedral angle of A/B = 2.78 (6)°. Atoms Br1, O1 and C9 are at distances of 0.0415 (4), 0.0464 (8) and −0.0244 (7) Å, respectively, from the best plane of the bromoindoline unit. The 1-{2-[2-(2-chloroethoxy)ethoxy]ethyl} moiety is oriented with respect to the bromoindoline unit by 77.7 (2)°, as defined by the C10—C9—N1—C1 torsion angle.The title compound, (I)Brmind⋯ODio, C—HBrmind⋯OEthy, C—HChlethy⋯ODio and C—HChlethy⋯OChlethy  hydrogen bonds (Table 1Cg1—Cg1i , may further stabilize the structure, with a centroid–centroid distance of 3.899 (2) Å. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (28.1%), H⋯O/O⋯H (23.5%), H⋯Br/Br⋯H (13.8%), H⋯Cl/Cl⋯H (13.0%) and H⋯C/C⋯H (10.2%) interactions. Hydrogen bonding and van der Waals interactions are the dominant interactions in the crystal packing.In the crystal, intermolecular C—Hs Table 1 link thefs Fig. 2. π–π conCrystalExplorer17.5  and those delineated into H⋯H, H⋯O/O⋯H, H⋯Br/Br⋯H, H⋯Cl/Cl⋯H, H⋯C/C⋯H, O⋯C/C⋯O, C⋯C and O⋯Cl/Cl⋯O contacts –(i), respectively, together with their relative contributions to the Hirshfeld surface. The most important interaction is H⋯H, contributing 28.1% to the overall crystal packing, which is reflected in Fig. 6b) as widely scattered points of high density due to the large hydrogen content of the molecule with the tip at de = di ∼1.08 Å, due to the short interatomic H⋯H contacts (Table 2c), with a 23.5% contribution to the HS, arises from the H⋯O/O⋯H contacts  and H⋯Cl/Cl⋯H  contacts, with 13.8 and 13.0% contributions to the HS, have nearly symmetrical distributions of points with the edges at de + di = 2.92 (for thin edge) and 3.20 Å (for thick edge) and de + di = 2.78 Å, respectively, arising from the H⋯Br/Br⋯H and H⋯Cl/Cl⋯H contacts (Table 2f), a 10.2% contribution to the HS, arises from the H⋯C/C⋯H contacts , with a 4.0% contribution to the HS, arises from the O⋯C/C⋯O contacts , with a 2.6% contribution to the HS, have a nearly arrow-shaped distribution of points arising from the C⋯C contacts  contacts, with a 1.1% contribution to the HS, have nearly symmetrical distributions of points with the edge at de + di = 3.50 Å.In order to visualize the intermolecular interactions in the crystal of the title compound, a Hirshfeld surface (HS) analysis –(e), respectively.The Hirshfeld surface representations with the function et al., 2015The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions suggest that van der Waals interactions and hydrogen bonding play the major roles in the crystal packing  using standard B3LYP functional and 6-311G basis-set calculations  of the molecule was about 6.5402 eV, and the frontier molecular orbital energies, i.e.EHOMO and ELUMO, were −7.4517 and −0.9115 eV, respectively.The optimized structure of the title compound, (I)et al., 2010et al., 2010H-indole-2,3-dione  and dimethylformamide  in potassium carbonate  and tetra-n-butylammonium bromide . The mixture was stirred at 353 K for 48 h. The reaction was controlled by CCM. The solution was filtered and the DMF was removed under vacuum. The product obtained was separated by chromatography on a column of silica gel with hexane–ethyl acetate (4:1 v/v) as eluent. The isolated solid was recrystallized from ethanol to afford red crystals .1,2-Bis(2-chloroethoxy)ethane  was added dropwise to a solution of 5-bromo-1Uiso(H) = 1.2Ueq(C). During the refinement process, the disordered chloroethoxyethoxyethyl side-chain atoms were refined with a major–minor occupancy ratio of 0.665 (8):0.335 (6).The experimental details, including the crystal data, data collection and refinement, are summarized in Table 310.1107/S2056989019011617/lh5913sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019011617/lh5913Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019011617/lh5913Isup3.cdxSupporting information file. DOI: 1948316CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structures of two first-row transition metal (Fe and Co) pyridine–sulfate complexes are presented. The compounds demonstrate infinite chains of metal pyridine units connected by bridging sulfate anions. catena-poly[[tetrakis(pyridine-κN)iron(II)]-μ-sulfato-κ2O:O′], [Fe(SO4)(C5H5N)4]n, (1), and catena-poly[[tetrakis(pyridine-κN)cobalt(II)]-μ-sulfato-κ2O:O′-[tetrakis(pyridine-κN)cobalt(II)]-μ-sulfato-κ3O,O′:O′′-[tris(pyridine-κN)cobalt(II)]-μ-sulfato-κ2O:O′], [Co3(SO4)3(C5H5N)11]n, (2), are reported. The iron compound (1) displays a polymeric structure, with infinite chains of FeII atoms adopting octahedral N4O2 coordination environments that involve four pyridine ligands and two bridging sulfate ligands. The cobalt compound (2) displays a polymeric structure, with infinite chains of CoII atoms. Two of the three Co centers have an octahedral N4O2 coordination environment that involves four pyridine ligands and two bridging sulfate ligands. The third Co center has an octahedral N3O3 coordination environment that involves three pyridine ligands, and two bridging sulfate ligands with one sulfate chelating the cobalt atom.The solid-state structures of two metal–pyridine–sulfate compounds, namely  The first reports of a pyridine–sulfato–metal complex were in the late 19th century  and the cobalt–pyridine–sulfate (2) complexes.Against this backdrop, our lab has recently begun to study the solid-state structures of transition-metal pyridine complexes. We have recently reported the structures of nickel, copper and zinc pyridine sulfates, which showed varying coordination geometries consistent with those predicted by crystal field theory (Roy 1), the asymmetric unit consists of two pyridine molecules and one half of a sulfate anion coordinated to an iron atom sitting on an inversion center . When grown out, the iron displays an octahedral coordination environment . There is a square-planar tetrapyridine iron unit, with FeN4 planarity enforced by the inversion. The octahedral coordination is completed by two sulfate ions that bind trans to each other. The cis N—Fe—N angles have values of 86.44 (4) and 93.56 (4)° and the cis O—Fe—N angles have values ranging from 88.12 (4) to 91.88 (4)°. The pyridine rings are rotated from the FeN4 plane by dihedral angles of 44.03 (1) and 78.20 (1)°. The 78.20 (1)° angle is constrained by two C—H⋯O interactions with the trans sulfates , the asymmetric unit consists of three cobalt atoms, eleven coordinated pyridine molecules, and three sulfate anions . There are three crystallographically independent cobalt atoms, with Co1  and Co2  displaying octahedral N4O2 coordination environments, and Co3 showing an octahedral N3O3 coordination environment .In the pink crystals of  to 93.21 (9)°, and the O—Co—O angle is 174.62 (9)°. The four pyridine rings are rotated from the CoN4 plane by dihedral angles of 37.51 (1), 45.21 (1), 56.40 (1) and 56.92 (1)°. Two of the rings form one C—H⋯O interaction each with the sulfate oxygen atoms  to 93.19 (9)°, and the O—Co—O angle is 175.16 (9)°. The four pyridine rings are rotated from the CoN4 plane by dihedral angles of 55.37 (1), 65.88 (1), 67.08 (1) and 68.07 (1)°. Two of the rings are involved in two C—H⋯O interactions each with the sulfate oxygen atoms  that form a CoO3 plane with a maximum deviation from planarity of 0.029 Å. The meridional CoN3 and CoO3 planes are rotated relative to one another by an angle of 88.93 (1)°. The cis N—Co—N angles have values of 86.76 (10) and 87.52 (9)°. The chelating sulfate exhibits an O—Co—O bite angle of 65.36 (7)° and another cis O—Co—O angle of 88.63 (8)°. The three pyridine rings are rotated from the CoN3 plane by dihedral angles of 31.855 (2), 44.111 (3) and 82.863 (4)°. The 82.863 (4)° angle is constrained by two C—H⋯O interactions with sulfate oxygen atoms , the FeII atoms are linked together into infinite chains along the [100] direction through the sulfate ligands via O—S—O bridges . Between each successive tetrapyridine iron unit are found parallel slipped π–π interactions .In compound , the CoII atoms linked together into infinite chains along the [001] direction through the sulfate ligands . No π–π interactions are observed in this crystal. There are two C—H⋯O interactions between chains  that connect the chains in three dimensions  and steric effects could play significant roles in determining the observed coordination environments.In a report earlier this year, we presented the structures of the metal–pyridine–sulfates of nickel, copper and zinc. It was of note that these three structures exhibited different coordination geometries, consistent with the crystal field stabilization energies (CFSE) associated with their Approximately 25 mg of each metal sulfate  were dissolved in pyridine  in a 20 mL vial under an atmosphere of dinitrogen. In the cobalt case, 0.1 mL of water was also added. The vials were heated to 353 K for 24–48 h, after which single crystals suitable for X-ray diffraction studies were isolated.SHELXL) by full-matrix least squares on F2. Hydrogen atoms were placed in calculated positions and then refined with a riding model with C—H bond lengths of 0.95 Å and with isotropic displacement parameters set to 1.20 Ueq of the parent C atom.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018007557/sj5556sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018007557/sj55561sup4.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989018007557/sj55562sup5.hklStructure factors: contains datablock(s) 2. DOI: 1844143, 1844142CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title structure has a square-planar coordination sphere around the copper(II) ion. In the crystal, molecules are linked by weak C—H⋯O and C—H⋯π hydrogen bonds and very weak π-stacking interactions, forming a three-dimensional supramolecular architecture. 18H12F6N2O4)]·0.5C6H6O2, the CuII ion has a square-planar coordination geometry, being ligated by two N and two O atoms of the tetradentate open-chain Schiff base ligand 6,6′-{-bis(methanylylidene)}bis[2-(trifluoromethoxy)phenol]. The crystal packing is stabilized by intramolecular O—H⋯O and intermolecular C—H⋯F, C—H⋯O and C—H⋯π hydrogen bonds. In addition, weak π–π interactions form a three-dimensional structure. Hirshfeld surface analysis and two-dimensional fingerprint plots were performed and created to analyze the intermolecular interactions present in the crystal, indicating that the most important contributions for the crystal packing are from F⋯H/H⋯F (25.7%), H⋯H (23.5%) and C⋯H/H⋯C (12.6%) interactions.In the title complex, [Cu(C In this study, we describe the crystal structure and Hirshfeld surface analysis of the title compound, as determined by X-ray crystallographic analysis.In this study, a salen-type Schiff base has been synthesized from 2-hydroxy-5-(trifluoromethoxy)benzaldehyde with ethylenediamine by a condensation reaction. The synthesized Schiff base was used as an II ion chelated by a doubly deprotonated tetradentate Schiff base ligand and a hydrogen-bonded molecule of hydroquinone. The Cu1 ion is coordinated by two imine N atoms (N6 and N7) and two phenoxo O atoms (O2 and O3) of the tetradentate Schiff base ligand 6,6′-{-bis(methanylylidene)}bis[2-(trifluoromethoxy)phenol] (L1). The hydroquinone molecule is located on an inversion centre and is linked to neighbouring complex cations via O—H⋯O hydrogen bonds. The bond lengths Cu1—O2 and Cu1—O3  and Cu1—N1 and Cu1—N2  are close to the values observed for related copper(II) complexes reported in the literature  hydrogen bonds (Table 2Cg2⋯Cg3 distance is 3.507 (2) Å, where Cg2 and Cg3 are the centroids of the Cu1/O2/C5/C6/C8/N2 and Cu1/O3/C17/C12/C11/N1 rings, respectively.The crystal packing of the title compound is stabilized by intermolecular C—H⋯O, C—H⋯F and C—H⋯s Table 2. In addis Table 2. The Cg2CrystalExplorer17 . The blue, white and red colour conventions used for the dnorm-mapped Hirshfeld surfaces recognize the interatomic contacts as longer, at van der Waals separations and short interatomic contacts, respectively.The Hirshfeld surface analysis  pair with the full fingerprint plot outlined in gray. Fig. 5a) shows the two-dimensional fingerprint plot of the sum of the contacts contributing to the Hirshfeld surface represented in normal mode. The most significant contribution to the Hirshfeld surface is from F⋯H/H⋯F contacts (25.7%) . Here, H⋯H interactions are only the second most significant contribution to the total Hirshfeld surface (23.5%). In addition, C⋯H/H⋯C and O⋯H/H⋯O contacts contribute 12.6 and 11.2% to the Hirshfeld surface, respectively.A fingerprint plot delineated into specific interatomic contacts contains information related to specific intermolecular interactions. The blue colour refers to the frequency of occurrence of the (%) Fig. 5b. Here,et al., 20163  and 1.929 (5) Å] fall within these limits. While the Cu1—O3 and C1—O2 bond length  are within and close to these limits, respectively, the Cu1—O2 bond length is outside these limits, with a shorter value of 1.883 (4) Å.A search of the Cambridge Structural Database -bis(methanylylidene)}bis[4-(trifluoromethoxy)phenol] (H2L1) was synthesized by condensation of 2-hydroxy-5-(trifluoromethoxy)benzaldehyde (0.0095 mmol) and 1,2-ethanediamine (0.0095 mmol) in ethanol under reflux for about 18 h. The yellow product was washed with ether and dried at room temperature. 0.0080 mmol H2L1 was dissolved in 20 ml ethanol and 0.0080 mmol Cu(CH3COO)2·H2O was dissolved in 20 ml ethanol. The metal solution was added dropwise to the Schiff base solution and the resulting solution refluxed for about 6 h. The product (CuL1) was washed with toluene and crystallized from ethanol at room temperature. 2,2′-{-bis(methanylylidene)}bis[4-(trifluoromethoxy)phenol]copper(II) hydroquinone hemisolvate was obtained even after 0.0040 mmol hydroquinone was added to 0.0040 mmol CuL1 in 20 ml ethanol and refluxed for about 6 h. A purple crystal suitable for X-ray diffraction analysis was obtained from the reaction   Fig. 6.Uiso(H) = 1.2Ueq(C) for methylene, C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C) for aromatic, C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C) for methine, and O—H = 0.82 Å and Uiso(H) = 1.5Ueq(O) for hydroxy H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019014294/lh5932sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019014294/lh5932Isup2.hklStructure factors: contains datablock(s) I. DOI: 1900670, 1900670CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title Schiff base compound was obtained from a condensation reaction of 4-chloro-3-hydroxybenzaldehyde and 2,4-dinitrophenylhydrazine. The molecule is almost planar with the dihedral angle between the benzene rings being 3.70 (17)°. 13H9ClN4O5·0.5CH3CN, crystallizes as an acetonitrile hemisolvate; the solvent molecule being located on a twofold rotation axis. The molecule is nearly planar, with a dihedral angle between the two benzene rings of 3.7 (2)°. The configuration about the C=N bond is E, and there is an intramolecular N—H⋯Onitro hydrogen bond present forming an S(6) ring motif. In the crystal, molecules are linked by O—H⋯O and N—H⋯O hydrogen bonds, forming layers lying parallel to (10The title Schiff base compound, C These compounds show biological activities including antibacterial, antifungal, anticancer and herbicidal activities  ring motif  Å, where Cg1 and Cg2 are the centroids of rings C1–C6 and C8–C13, respectively, α = 3.70 (17)°, β = 27.9°, γ = 24.5°, interplanar distances are 3.489 (2) and 3.388 (2) Å, offset = 1.791 Å; symmetry code: (i) −x + 1, −y + 1, −z + 1. There are no other significant intermolecular contacts present in the crystal.In the crystal, molecules are linked by O—H⋯O and N—H⋯O hydrogen bonds Table 1, formingGAUSSIAN09  level . 18 of these structures involve a halide substituent and 23 involve a hydroxyl substituent. Only one compound involves both a halide and an hydroxyl substituent and closely resembles the title compound, viz. 4-chloro-2-{[hydrazono]methyl}phenol  ring motif. In fact, in all 71 structures (see supporting information) there is an intramolecular N—H⋯Onitro hydrogen bond present forming an S(6) ring motif, and in the majority of the compounds the two benzene rings are almost coplanar with the dihedral angle varying between ca 0 to 8°, with a few exceptions.A search of the Cambridge Structural Database  in ethanol (15 ml) and 2,4-dinitrophenylhydrazine  in ethanol (15 ml). The reaction mixture was stirred for 5 h under reflux. Orange plate-like crystals of the title compound were obtained by slow evaporation of a solution in ethanol .Uiso(H) = 1.5Ueq and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901900642X/su5496sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S205698901900642X/su5496Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698901900642X/su5496sup3.pdfCSD search S1. DOI: Click here for additional data file.10.1107/S205698901900642X/su5496Isup4.cmlSupporting information file. DOI: 1912273CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title chalcone derivative is almost planar, with a dihedral angle of 7.0 (2)° between the 3,5-dichloro-2-hydroxyphenyl and 5-methylfuran rings. 14H10Cl2O3, is almost planar, with a dihedral angle of 7.0 (2) ° between the 3,5-dichloro-2-hydroxyphenyl and 5-methylfuran rings. There is an intramolecular O—H⋯O hydrogen bond present forming an S(6) ring motif. In the crystal, molecules are linked by bifurcated C—H/H⋯O hydrogen bonds, enclosing an R12(6) ring motif, forming a 21 helix propagating along the b-axis direction. The intermolecular interactions were quantified using Hirshfeld surface analysis.The title chalcone derivative, C They belong to the flavonoid family, which are basically found in fruits and vegetables -1--3-(5-methylfuran-2-yl)prop-2-en-1-one.Chalcone derivatives are an important class of organic compounds comprising two aromatic rings connected via an unsaturated α,β carbonyl system as shown in Fig. 1S(6) ring motif (Table 1anti-periplanar conformation described by the torsion angles C11—C12—C13—Cl19 = −179.1 (3)° and C13—C14—C15—Cl18 = −178.6 (4)°, while methyl group at C2 of the furan ring is in a +anti-periplanar conformation [C5—O1—C2—C6 = 178.3 (4)°]. The bond lengths and angles in the title compound are similar to those observed for 3-(furan-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one  Å, propagating along the b-axis direction  < 1.8 Å (di is the distance of a point on the Hirshfeld surface to the nearest nucleus inside the surface while de is the distance of the nearest nucleus outside the surface). The H⋯H contacts, with a contribution of 25.7%, are shown as blue dots spread in the middle region 1.18 Å < (de + di) < 1.62 Å. The two sharp spikes observed at 1.04 Å < (de + di) < 1.39 Å are due to the presence of a pair of O⋯H contacts making a 15.2% contribution. A pair of C⋯H contacts are observed as characteristic wings in the region of 1.18 Å < (de + di) < 1.6 Å (13.0% contribution). C⋯C, C⋯Cl and O⋯C contacts make contributions of 7.9%, 5.2% and 3.8%, respectively.The two-dimensional fingerprint Fig. 6 plots weet al., 2016et al., 2015et al., 2014et al., 2014S(6) ring motifs. The molecules are all relatively planar with the dihedral angle between the furan and 2-hydroxyphenyl rings varying from ca 8.35° in BOGVID, 0.20° in KUDMON, and 10.90 and 2.56° in the two independent molecules of POJBAT. The only exception is POHZUJ, which possesses twofold rotation symmetry and has two [3-(2-furyl)prop-2-en-1-one] units meta to each other; here the dihedral angle is ca 19.87°.A search of the Cambridge Structural Database -2-hydroxyethanone (5 mmol) was dissolved in methanol (15 ml) and was stirred with 5 ml of sodium hydroxide solution for 30 min at room temperature. To this mixture, 5-methylfuran-2-carbaldehyde (5 mmol) was added over 30 min with stirring. Stirring at room temperature was then continued for 32 h. On completion of the reaction, monitored by TLC, the mixture was quenched in ice–water and acidified with dilute hydrochloric acid. The separated precipitate of the title compound was filtered off and recrystallized from methanol solution giving colourless block-like crystals.Uiso(H) = 1.2Ueq(C) for aromatic H atoms and C—H = 0.96 Å with Uiso(H) = 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018012173/qm2127sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018012173/qm2127Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018012173/qm2127Isup3.cmlSupporting information file. DOI: 1852049CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


WC)], reverse Watson-Crick [A·T(wrWC)], Hoogsteen [A·T(wH)] and reverse Hoogsteen [A·T(wrH)] – act as intermediates of the intrapair mutagenic tautomerization of the T nucleobase owing to the novel tautomerisation pathways: A·T(wWC)↔A·T*(w⊥WC); A·T(wrWC)↔A·⊥rWC); A·T(wH)↔A·T*(w⊥H); A·T(wrH)↔A·⊥rH). All of them occur via the transition states as tight ion pairs · with quasi-orthogonal geometry, which are stabilized by the participation of the strong (A)N6+H···O4−/O2−(T) and (A)N6+H···N3−(T) H-bonds. Established tautomerizations proceed through a two-step mechanism of the protons moving in the opposite directions along the intermolecular H-bonds. Initially, proton moves from the N3H imino group of T to the N6H2 amino group of A and then subsequently from the protonated N6+H3 amino group of A to the O4/O2 oxygen atom of T, leading to the products – A·T*(w⊥WC), A·⊥rWC), A·T*(w⊥H), and A·⊥rH), which are substantially non-planar, conformationally-labile complexes. These mispairs are stabilized by the participation of the (A)N6H/N6H'···N3(T) and (T)O2H/O4H···N6(A) H-bonds, for which the pyramidalized amino group of A is their donor and acceptor. The Gibbs free energy of activation of these mutagenic tautomerizations lies in the range of 27.8–29.8 kcal·mol−1 at T = 298.15 K in the continuum with ε = 1.In this paper using quantum-mechanical (QM) calculations in combination with Bader's quantum theory of “Atoms in Molecules” (QTAIM) in the continuum with ε = 1, we have theoretically demonstrated for the first time that revealed recently highly-energetic conformers of the classical A·T DNA base pairs – Watson-Crick [A·T(w Protonated amino group N6+H3 of A for these TSs acts simultaneously as donor and acceptor of the H-bonding and has such spatial orientation, that its N6+H/N6+H′ bond, which is not involved in the H-bonding with T, lies in the plane of the purine ring R, L (10.44), A·T*(w⊥H)R, L (14.69), A·⊥rWC)R, L (9.20) and A·⊥rH)R, L (13.75) kcal·mol−1 complexes (C1 symmetry), which are the products of these mutagenic tautomerizations, are stabilized by the two anti-parallel (T)O4H/O2H··· N6(A) (~5.5) and (A)N6H/N6H′··· N3(T)  H-bonds R, L 0.44, A·T⊥WC)R, L 0.44, A·Tcys/trans mutual orientation of the N1H and N9H glycosydic bonds of the bases. At the mutagenic tautomeric transformations of the DNA bases some R/L structures transfer into the other L/R structures and vice versa  and −1) with low values of the imaginary frequencies provided in the brackets. At this, one-single intermolecular (T)O4H/O2H··· N6(A) H-bond between the O4H/O2H hydroxyl groups of T*/1s. In the case of TS2s, when T hangs over A, the (T)O4H/O2H··· N6(A) H-bond coexists together with attractive van der Waals contacts with significantly increased ellipticity – N3··· C6 and O2··· C4 in the case of 1s are the most energetically favorable (1.86 and 1.92) in comparison with the TS2s  (Table Two of these tautomerization reactions are controlled by the TSs – vice versa. In these cases TSs – −1) and −1) are characterized by the considerably higher values of imaginary frequencies  and stabilized by two antiparallel (T)O4/O2H··· N6(A) and (A)N6H/N6H′··· N3(T) H-bonds, the first of which is significantly stronger, then the other one  contribute only a part of the electron energy of the monomer interactions R,L↔ A·T*(w⊥H)R,L and A·T*O2(*O2(w⊥rH)R,L, tautomerizations indicating the close structural relationship between tautomerization the classical A·T(WC) and A·T(H) DNA base pairs, on the one hand, and A·T(rWC) and A·T(rH), on the other hand ], reverse Watson-Crick [A·T(wrWC)], Hoogsteen [A·T(wH)] and reverse Hoogsteen [A·T(wrH)], which have been analyzed in details in our previous paper ↔A·T*(w⊥WC), A·T(wrWC)↔A·⊥rWC), A·T(wH)↔A·T*(w⊥H), A·T(wrH)↔A·⊥rH) – proceed through the stepwise proton transfer via the TSs as tight A+·T− ion pairs, which Gibbs free energy of activation lies in the range of 27.79–29.83 kcal·mol−1 at T = 298.15 K, thus creating the substantially non-planar, conformationally-labile complexes – A·T*(w⊥WC), A·⊥rWC), A·T*(w⊥H) and A·⊥rH). Furthermore, formed complexes involving mutagenic T*/*(w⊥WC)↔A·T*(w⊥H) and A·⊥rWC)↔A·⊥rH).Here we have shed light on the revealed for the first time physico-chemical mechanism of the intrapair mutagenic tautomerization of the T DNA base within the novel highly-energetic conformers of the classical A·T DNA base pairs – Watson-Crick [A·T(wOB, study conception and design, acquisition of data, drafting of manuscript analysis and interpretation of data, performance of calculations, discussion of the obtained data, preparation of the numerical data for Tables, graphical materials for Figures and text of the manuscript. KT, preparation of the numerical data for Tables and graphical materials for Figures, preparation of the text of the manuscript. AD, analysis and preparation of the current literature survey, discussion of the strategy of the current investigation, analysis of the obtained numerical data, discussion of the obtained data, preparation of the numerical data for Tables, graphical materials for Figures and text of the manuscript. DH, study conception, critical revision of manuscript, proposition of the task of the investigation, discussion of the obtained data, preparation of the text of the manuscript. All authors were involved in the proofreading of the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.A high‐throughput method (≥ 10 Enzyme‐based organic synthesis constantly needs innovative, diverse and robust biocatalysts  construction of metagenomic libraries, and subsequent functional screening  or by using tributyrin‐supplemented agar plates ‐ and (S)‐enantiomers has been used as a substrate in the bond‐breaking reaction that generates a growth‐promoting energy source and a growth‐inhibiting compound  and were omitted from the further analysis. The plasmid DNA from the remaining clones was isolated, and the fragments obtained after restriction digestion were analysed by sequencing to exclude the redundancy. Hence, 30 clones were chosen for further analysis.The metagenomic DNA isolated from different soil samples was partially digested with several restriction endonucleases, and the fragmented DNA was used for the construction of metagenomics libraries. In total, 19 libraries  were tesBioinformatics analysis showed that the selected clones exhibiting esterase activity contained ORFs with medium 31%) to high (100%) sequence identity to proteins found in the databases (Table % to highN‐acyltransferase superfamily (SSF55729) (1 hit) and one DUF998 family protein  revealed that the enzyme 1315H may be a transmembrane protein without any predictable function.The phylogenetic analysis of the selected hydrolases showed that the enzymes represent very diverse groups of proteins Fig. . Most ofein Fig. . All ideein Fig.  and wereein Fig. . Two estThus, a functional selection of metagenomic libraries revealed enzymes from very diverse protein families, including several hits that, based on bioinformatics, could not be annotated as hydrolases active towards esters.E. coli strain BL21 (DE3) was transformed with the recombinant plasmids and used for the expression of the recombinant proteins. In total, 27 recombinant proteins were purified by Ni‐NTA chromatography  esters: acetate, butyrate, valerate, decanoate, palmitate and stearate. All hydrolases were active towards the short‐chain esters pNP‐acetate and pNP‐butyrate, most of them used pNP‐valerate, but only a few of them hydrolyzed pNP‐decanoate  exhibited a very weak activity against this ester. Six enzymes MO101T, SVG3, GRU1, PLA1, 3T and 33T3 exhibited a strong preference for pNP‐acetate as the substrate  forming the halos indicative of the hydrolysis were screened. Tb7_1T (MH423281) encoded the group β‐lactamases protein, and Tb10_7T (MH423280) was most similar to ABhydrolases. Neither of the two clones was capable of growing on compound 1 or 2 as a uridine source. Tb7‐1T was not further analysed because of its insolubility. The purified protein Tb10_7 was not only active towards pNP esters  and two  esterases preferred S‐1‐phenylethyl hexanoate and S‐1‐phenylethyl benzoate respectively. No esterase was enantioselective towards R‐1‐phenylethyl hexanoate or benzoate. Furthermore, to analyse the promiscuity of the selected esterases, several amides were tested as substrates. Consequently, three esterases were found to hydrolyze acetanilide, and 18 were active towards nitrocefin  and the uridine auxotroph mutant ΔpyrFEC of E. coli allows for a functional selection of novel ester hydrolases from metagenomic libraries. Evidently, the selection method presented here is highly complementary to traditional approaches and is applicable for allowing the discovery of novel esterases with different structural and catalytic characteristics. Compared with the known methods, the proposed selection system has many advantages: (i) it is a HTS method that allows for a rapid (1–4 days) processing of large (meta)genome libraries  with low number of false positives, ii) depending on the selection media used, it permits the functional selection of hydrolases belonging to different protein families, (iii) it is flexible since, by using different substrates, for example, esters of the fatty acids of different lengths or bulky carboxylic acids, it allows the identification of enzymes with desired properties, (iv) it would be suitable for the selection of regioselective esterases if the appropriate substrate was used, (v) it allows the identification of novel proteins that previously have not been known as the ester‐hydrolysing enzymes or cannot be screened using, for example, a tributyrin method, (vi) the appropriate uridine auxotrophs are available for a wide spectra of microbial hosts, including extremophiles and yeasts or can be easily constructed, for example, using 5‐fluororotic acid selection  Assay Reagent and HisPur™ Ni‐NTA spin column were purchased from Thermo Fisher Scientific, Rockford, IL. pET21a Vector was purchased from Novagen . Nutrient medium were purchased Roth, Germany. pNP‐acyl esters, tributyrin, β‐d‐glucose pentaacetate, β‐d‐galactose pentaacetate, and uridine were purchased from Sigma‐Aldrich. ‘ZR Soil Microbe DNA MidiPrep™’ was purchased from Zymo Research, Freiburg, Germany. Nitrocefin was purchased from Oxoid, UK. 3′‐O‐benzoyl‐2′‐deoxyuridine were purchased from Carbosynth, UK. 3′‐O‐acetyl‐2′‐deoxyuridine, 3′‐O‐acetyl‐N4‐benzoyl‐2′‐deoxycytidine, 3′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine, and 5′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine were purchased from Jena Bioscience, Jena, Germany.Restriction endonucleases, HindIII, BamHI, PstI, Phusion DNA polymerase, aLICator™ LIC Cloning and Expression System Kit 3, PageRulerO‐acetyluridine and 2′,3′,5′‐hexanoyluridine were prepared by adapting and modifying methods reported in the literature and evaporated under reduced pressure. The residue was purified by column chromatography  to afford 2′,3′,5′‐O‐triacetyluridine (1.45 g (95%) as a white solid. UV (CH3OH) λmax 257 nm. MS (ESI+): m/z 371.10 [M+H]+, 369.10 [M−H]−. 1H‐NMR (DMSO‐d6): δ = 2.11 , 2.13 , 2.15 , 4.36 , 5.35 , 5.81 , 6.05 , 7.41 , 9.69 . 13C‐NMR (DMSO‐d6): δ = 20.41, 20.51, 20.77, 63.18, 70.20, 72.72, 79.92, 87.46, 103.44, 139.36, 150.35, 163.00, 168.75, 169.67, 170.18. The NMR spectra were consistent with reported previously  and sodium acetate  was heated for 20 min at 90°C. Then uridine  was added to the hot solution and stirred for 1 h. After the reaction was completed (TLC), the mixture was quenched with sodium bicarbonate and extracted with chloroform. The organic phase was dried  and N,N‐dimethylpyridin‐4‐amine  were added. The reaction mixture was stirred for 3 h at room temperature. After the reaction was completed (TLC), the formed precipitate of N,N′‐dicyclohexylurea was filtered, and the filtrate was concentrated under reduced pressure. The residue was treated with acetonitrile and the mixture of mono‐, di‐ and tri‐O‐hexanoyluridines precipitated as a white solid. The precipitate was filtered, dried to afford 0.5 g of hexanoyluridines. HPLC‐MS analysis indicated that the mixture consisted of 7% of mono‐O‐hexanoyluridine, 21% of di‐O‐hexanoyluridine and 72% of tri‐O‐hexanoyluridine. UV (CH3OH) λmax 256 nm. MS (ESI+): m/z 341.00 [M−H]−, 439.05 [M−H]−, 537.10 [M−H].To a stirred mixture of uridine  and triethylamine  in 1,4‐dioxane (10 ml) hexanoic acid , R)‐1‐phenylethanol or (S)‐1‐phenylethanol  and DMAP  were dissolved in acetic anhydride  and heated for 30 min at 55°C. The reaction mixture was monitored by thin‐layer chromatography . After the reaction was completed, the formed acetic acid was evaporated under reduced pressure. The residue was purified by column chromatography . After removal of solvents in vacuum, the desired products were obtained as colourless oils.Enantiomerically pure (Rf = 0.68 (CHCl3).1H NMR (CDCl3): δ = 1.57 , 2.10 ; 5.92 , 7.27–7.44 . 13C NMR (CDCl3): δ = 21.37, 22.22, 72.32, 126.11, 127.88, 128.51, 141.69, 170.34.Yield 261 mg (97%), colourless oil, Rf = 0.69 (CHCl3).1H NMR (CDCl3): δ = 1.57 , 2.10 ; 5.91 , 7.27–7.44 . 13C NMR (CDCl3): δ  =  21.37, 22.22, 72.32, 126.10, 127.88, 128.51, 141.69, 170.35.Yield 219 mg (81%), colourless oil, (R)‐1‐phenylethyl acetate reported in literature ‐1‐phenylethanol or (S)‐1‐phenylethanol  and triethylamine  were dissolved in dichloromethane (2 ml). After the solution was cooled to an ice bath, benzoyl chloride  was added, and the reaction mixture was stirred for 1 h at ambient temperature. After the reaction was completed (TLC), the solvent was evaporated under reduced pressure. The residue was purified by column chromatography . The solvents were removed in vacuum, and the desired products were obtained as colourless oils.Enantiomerically pure (Rf = 0.75 (CHCl3).1H NMR (CDCl3): δ = 1.71 , 6.17 , 7.31–7.52 , 8.10–8.16 . 13C NMR (CDCl3): δ = 22.45, 72.94, 126.07, 127.92, 128.36, 128.58, 129.67, 130.54, 132.95, 141.81,165.84.Yield 184 mg (50%), colourless oil, Rf = 0.76 (CHCl3).1H NMR (CDCl3): δ = 1.71 , 6.17 , 7.31–7.53 , 8.09–8.15 . 13C NMR (CDCl3): δ = 22.44, 72.93, 126.07, 127.92, 128.36, 128.57, 129.67, 130.55, 132.95, 141.81,165.84.Yield 360 mg (97%), colourless oil, 1H NMR spectrum was consistent with spectrum of 1‐phenylethyl benzoate racemic ester reported previously ‐1‐phenylethanol or (S)‐1‐phenylethanol , hexanoic acid , DCC  and DMAP  were dissolved in 10 ml of dichloromethane and stirred for 3 h at ambient temperature. After the reaction was completed (TLC), the formed precipitate was filtered, and the solvent was removed under reduced pressure. The residue was purified by column chromatography . After removal of solvents in vacuum, the desired products were obtained as colourless oils.Enantiomerically pure (Rf = 0.82 (CHCl3).1H NMR (CDCl3): δ = 0.91 , 1.25–1.42 , 1.56 , 1.61–1.70 , 2.33–2.38 , 5.93 , 7.28–7.41 . 13C‐NMR (CDCl3): δ = 13.91, 22.28, 22.32, 24.67, 31.27, 34.60, 72.01, 126.07, 127.79, 128.47, 141.88, 173.11.Yield 288 mg (80%), colourless oil, Rf = 0.82 (CHCl3).1H NMR (CDCl3): δ = 0.91 , 1.27–1.40 , 1.56 , 1.61–1.70 , 2.32–2.38 , 5.93 , 7.28–7.42 . 13C‐NMR (CDCl3): δ  =  13.91, 22.28, 22.32, 24.67, 31.27, 34.60, 72.01, 126.07, 127.79, 128.47, 141.88, 173.11.Yield 272 mg (75%), colourless oil, et al., NMR spectra were consistent with spectra of 1‐phenylethyl hexanoate racemic ester reported in literature  supplemented with the appropriate antibiotics , 1 mM IPTG and 1 mM X‐gal. Ten to twenty of individual white colonies‐forming clones were randomly chosen for plasmid DNA isolation by Thermo Scientific™ GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and analysis of the length of the insert. The remaining undiluted mixture of the cells was spread on LB agar ) supplemented with 40 μg ml−1 kanamycin and 100 μg ml−1 ampicillin. After cultivation for 14–16 h, a biomass was scraped from agar, and total plasmid DNA was isolated by Thermo Scientific™ GeneJET Plasmid Midiprep Kit (Thermo Fisher Scientific). The obtained DNA mixture (a metagenomics library) was stored at –20°C and used for a further transformation of the E. coli DH10B ΔpyrFEC::Km cells.Metagenomic libraries were constructed from soil and sediment samples using a pUC19 vector . For DNA−1 Na2HPO4 15 g l−1 KH2PO4, 5 g l−1 NH4Cl, 2.5 g l−1 NaCl, 2% (w/v) glucose, 0.2% Casamino acid, 1 mM IPTG) containing 100 μg ml−1 ampicillin and 40 μg ml−1 kanamycin, 0.02 mg ml−1 2′,3′,5′‐O‐acetyluridine (1) or 2′,3′,5′‐O‐hexanoyluridine (2) as the sole source of uridine, allowing only the growth of recombinants that can complement the uridine auxotrophy of the E. coli DH10B ΔpyrFEC::Km strain by hydrolysing the uridine esters.The clones exhibiting acetylesterase activity were identified on a MD medium . For further analysis the ORFs encoding putative hydrolases were chosen. When homology search did not predict a hydrolase, the deletion analysis to obtain the truncated variants of the plasmid and the functional reselection on the appropriate substrate was carried out. To confirm that the hits encoded the enzymes with an esterolytic activity, the selected genes were PCR‐amplified, and the resulting fragments were ligated into pET21a or pLATE31 expression vectors. Phylogenetic analysis was conducted using the Maximum Likelihood Tree routine of mega 7 software. and using the following sequencing primers: M13F‐pUC (5′‐GTTTTCCCAGTCACGAC‐3′), M13R‐pUC (5′‐CAGGAAACAGCTATGAC‐3′), T7 Promoter (5′‐ TAATACGACTCACTATAGGG‐3′), T7 terminator (5′‐TAATACGACTCACTATAGGG‐3′) or LIC Reverse Sequencing primer, 24‐mer (5′‐GAGCGGATAACAATTTCACACAGG‐3′). Some individual clones contained more than one ORF in each DNA fragment. ORFs were analysed by using the Unipro UGENE program, and homology search was conducted using the Blast server (E. coli cells transformed with recombinant plasmids were cultivated on LB agar supplemented with 100 μg ml ampicillin.The genes of the selected enzymes were amplified with Phusion DNA polymerase using primers . MetagenE. coli strain BL21 (DE3). E. coli cells were grown in 20 ml BHI (Brain‐Heart‐Infusion Broth) medium containing ampicillin (100 mg ml) at 37°C with aeration. Protein expression was induced by adding 0.5 μM IPTG at 0.6–1 OD600, and cells were grown for a further 4–18 h at 30°C. Cells expressing 12T and 3T were grown at 23°C after induction to increase protein solubility. Wet cell biomass from 20 ml culture broth were suspended in 5–10 ml of buffer A  and disrupted by sonication for 2.5 min. A lysate was cleared by centrifugation at 15 000 × g for 4 min. Cleared lysate was applied to 0.2 ml HisPur™ Ni‐NTA spin column (equilibrated with the buffer A). The column was washed with the buffer A, and the proteins were eluted with buffer A containing 300 mM imidazole. The active fractions were combined and dialyzed against the buffer B , at 25°C. All the purification procedures were performed at room temperature.The recombinant proteins were overexpressed in The concentration of protein was determined using Pierce™ Coomassie Plus (Bradford) Assay Reagent by Standard Microplate Protocol. Proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis  according to Laemmli. Gels were developed in Coomassie Brilliant Blue G‐250 dye, scanned in 16 bit format and quantified by GelAnalyser program. Each sample contained 2 μg of total protein. Quantities of impurities and target proteins were estimated using calibration curve generated from known amounts of BSA: 0.125, 0.25 and 0.5 μg per band. The purity of analysed protein was calculated as the ratio between quantity of target protein and quantity of all proteins.et al., et al., ε = 12.3 M−1 cm−1) per minute. Enzyme activity was tested against different pNP‐acyl esters .The activity of esterase were assayed by incubating the enzyme with 1 mM pNP‐substrate (from 10 mM stock in DMSO) in 50 mM potassium phosphate, pH 7.5, buffer at a 37°C for 10 min in 100 μl reaction volume. 5–300 ng of protein, depending on the enzyme specificity for the substrate, were present into the reaction mixture. The absorbtion of the reaction mixture at 405 nm was measured against enzyme‐free blank to compensate for the substrate auto‐hydrolysis , pentaacetylglucose or pentaacetylgalactose (from 100 mM stock in acetone), 3′‐O‐acetyl‐2′‐deoxyuridine, 3′‐O‐acetyl‐N4‐benzoyl‐2′‐deoxycytidine, 3′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine, and 5′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine (100 mM stock in DMSO). The total reaction mix volume was 100 μl. Reaction mixture was incubated at room temperature up to 5 h. A change of colour from red to yellow indicated the hydrolysis of esters , 1 μl enzyme (0.1–4.6 μg/reaction) and 10 mM substrate: d‐glucose pentaacetate, β‐d‐ galactose pentaacetate (from 100 mM stock in acetone), 3′‐O‐benzoyl‐2′deoxyuridine (10 mg ml−1 stock in DMF), 3′‐O‐levulinyl‐N4‐benzoyl‐2′deocytidine and 5′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine (from 100 mM stock in DMSO). The total reaction mix volume was 20 μl. Reaction mixture was incubated at 30°C temperature up to 3 h. Thin‐layer chromatography (TLC) was conducted on the Merck silica gel 60F254 plates, using the dichloromethane and methanol (9:1) mixture of solvents. β‐d‐glucose pentaacetate and β‐d‐ galactose pentaacetate were visualized by anisaldehide stain . The plate was developed by heating on a hot plate. Synthetic nucleosides were exposing to UV light.A hydrolytic activity was assayed in reaction mixture containing 45 mM potassium phosphate buffer, pH 7.5, 1 μl enzyme (0.1–4.6 μg/reaction) and 10 mM substrate: β‐There are no conflicts to declare.Table S1. Metagenomic libraries used in this work.Table S2. Primers used for amplification of genes of selected hydrolases.Table S3. Predicted signal peptide sequences identified in the selected esterases by bioinformatics.Fig. S1. Selected clones on MD medium, MD+ compound 1, MD+compound 2 and MD+uridine after 2 days of incubation at 37°C.Fig. S2. The alignment was performed by ClustalW software.Fig. S3. Analysis of the purified esterases by SDS‐PAGE.Fig. S4. LB medium with tributyrin plates after 2 days of incubation at 37°C.Scheme S1. Synthesis of optically active esters (3–8).Click here for additional data file.


I ion is coordinated by two heterocyclic N atoms from two ligands in a linear configuration, forming a discrete coordination complex. There is an O—H⋯O hydrogen bond between 2-tza− and 2tzaH of adjacent complexes. The hydrogen atom is shared between the two oxygen atoms.The Ag 4H2NO2S)(C4H3NO2S)] or [Ag(2-Htza)(2-tza)] is reported . The AgI ion is coordinated by two heterocyclic N atoms from two ligands in a linear configuration, forming a discrete coordination complex. There is an O—H⋯O hydrogen bond between 2-tza− and 2tzaH of adjacent complexes. The hydrogen atom is shared between the two oxygen atoms. This interaction produces a hydrogen-bonded tape parallel to the [110] direction, which is augmented through intermolecular C—H⋯O hydrogen-bonding interactions between the bound thiazole groups. There is a further rather long Ag⋯O interaction  that assembles these tapes into columns, between which there are C—H⋯π interactions, leading to the formation of a three-dimensional supramolecular architecture.The linear two-coordinate silver (I) complex [Ag(C The whole molecular structure can be generated by an inversion centre; the AgI atom is located at the 2a Wyckoff position (I centre shows a linear coordination with two 2-tza ligands coordinating through the heterocyclic N atoms with an Ag—N bond length of 2.1463 (14) Å. Statistically one of these ligands has an appended carboxylic acid and the other a carboxylate. A rather long Ag⋯O2 interaction is also observed with the distance of 2.8401 (13) Å. This is significantly larger than the mean value [2.54 (11) Å] of the Ag⋯O=C distances in the Cambridge Database (2-tza−). The carboxylate is located close to a second symmetry-equivalent carboxylate generated by the symmetry operation 1 − x, −y, 1 − z. Statistically, one of these two groups is protonated. The close approach facilitates the formation of a linear hydrogen bond between them  of an adjacent discrete molecule (Table 1a). This tape is inclined at an angle of 55.278 (19)° with the (001) plane. Each tape is connected through an Ag⋯O1 interaction [d(Ag⋯O1) = 2.9606 (14) Å], generating columns of complex molecules along the [100] direction . These columns are further linked via C—H⋯π interactions between the thiazole rings  and 2-Htza  were dissolved in 5.0 mL of deionized water in a small vial (ca 16 mm in diameter). The vial was left undisturbed at ambient temperature for three days during which colourless block-shaped crystals of the title compound crystallized and were isolated for X-ray data collection.AgNOUiso(H) = 1.5Ueq(O). The C-bound H atoms were refined isotropically, with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019000124/nk2248sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019000124/nk2248Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019000124/nk2248Isup3.molSupporting information file. DOI: 1888609CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Polynitrile anions are known for their ability to combine with transition metals and co-ligands to form ternary systems. Here we report on the crystal structure of tris(2–2′-bipyridine)cobalt(II) bis. 10H8N2)3](C9H5N4O)2, the triscobalt(II) dication lies across a twofold rotation axes in the space group C2/c. The N atoms of the three bipyridine ligands form a distorted octahedron around the cobalt ion. All the N atoms of the polynitrile 1,1,3,3-tetracyano-2-ethoxypropenide anions participate in C—H⋯N hydrogen bonds ensuring crystal cohesion and forming a three-dimensional structure. The structure is further stabilized by C—H⋯π(cation) and anion⋯π(cation) interactions.In the title compound, [Co(C The cobalt ion is ligated by the N atoms of the 2,2′-bipyridine ligands forming a slightly distorted octahedral coordination sphere; the Co1—N bond lengths vary from 2.122 (3) to 2.148 (3) Å. In the bpy  unit, the pyridine rings are inclined to each other by 10.40 (16)°, while in the other bpy unit (involving atom N3) bisected by a twofold rotation axis the pyridine rings are coplanar. The observed distortion of the CoIIcoordination sphere is probably the consequence of the hydrogen bonding between the [Co(C10H8N2)3]2+ cation and the flexible tcnoet− anion (see Supramolecular features).The asymmetric unit of the title compound (I)− anion, the six central C—C distances within the anion range from 1.382 (4) to 1.429 (5) Å while the C≡N distances vary from 1.138 (4) to 1.151 (4) Å °.In the tcnoetÅ Table 1. As obseThe crystal packing of (I)− anions linked by eight C—H⋯N hydrogen bonds as shown in Fig. 3− anions are doubly connected to the cationic units  via C8—H8⋯N7iii; C11i—H11i⋯N6iii and their symmetric C8i—H8i⋯N7iv; C11—H11⋯N6iv. Four tcnoet− anions are linked to atoms N4 and N5  via C7—H7⋯N4iv, C2—H2⋯N5v, C7i—H7i⋯N4ii and C2i—H2i⋯N5vii. One of the anions plays the role of a donor in the structural linkage. Indeed, one tcnoet− anion is linked by an N⋯H—C interaction to the same [Co(C10H8N2)3]2+ unit (via N6⋯H11iii—C11iii and N7⋯C8ii—H8ii) and to two other cationic units by N4⋯H7i—C7i and N5⋯H2iv—C2iv interactions. This environment where the negative charge is delocalized over the central propenide unit as well as into the cyano groups is illustrated in Fig. 4The cations are surrounded by six tcnoetB⋯Cg1vi; see Table 2Cg2; see Table 2The crystal structure of (I)et al., 2016− anion associated with an organic cation to form a salt-like compound  the water molecule links the tcnoet− anion and the iron aggregate via O—H⋯N hydrogen bonds, forming chains, whereas in (I)ca 28.1° compared to 31.7 (3)° in (I)A search in the Cambridge Structural Database , 2,2-bipyridine  and K(tcnoet)  in water–ethanol . This mixture was sealed in a Teflon-lined autoclave and held at 423 K for three days, and then cooled to ambient temperature at a rate of 10 K h−1 (yield: 54%). Colourless plate-like crystals of the title compound were selected directly from the synthesized product.The title compound was synthesized solvothermally under autogenous pressure from a mixture of CoNOUiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018018261/su5465sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989018018261/su5465Isup2.hklStructure factors: contains datablock(s) I. DOI: 1887084CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity‐based probes to visualize catalytic pocket occupancy of candidate off‐target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α‐glucosidase were identified as major off‐targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD‐like models.Glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html), nonlysosomal β‐glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html); cytosolic β‐glucosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/21.html); α‐glucosidases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/20.html); β‐glucuronidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/31.html).Glucocerebrosidase ( In vivo target engagement of mechanism‐based glucocerebrosidase (GBA) inhibitors—conduritol B epoxide (CBE) and cyclophellitol (CP)—were examined in cultured cells, zebrafish larvae and mice by competitive activity‐based protein profiling (ABPP). This method utilizes suicide fluorescent enzyme reporter molecules to assess active site occupancy of target glycosidases by inhibitors. The in vivo targets of CBE and CP and their selectivity towards GBA were revealed. ABPactivity‐based probeABPPactivity‐based protein profilingCBEconduritol B epoxideCPcyclophellitoldpfdays postfertilizationGBAglucocerebrosidaseGDGaucher diseaseGlcSphglucosylsphingosinePDParkinson's diseasehttp://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html) is a retaining β‐glucosidase that degrades the glycosphingolipid, glucosylceramide. Inherited deficiencyof GBA is the cause of autosomal recessive Gaucher disease (GD) The lysosomal enzyme glucocerebrosidase  Fig. A 7, 8, 9m to 100 mm CBE for 2 h up to 60 days −1 body weight during 2 h up to 36 days in vitro inhibition of retaining α‐glucosidases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/20.html) in vitroin situhttp://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html), and inhibition of the lysosomal β‐glucuronidase  in mice in vivo towards GBA2 and other glycosidases is unknown.A major advantage of CBE's pharmacological use in cultured cells and mice is its tunability: the extent of GBA inactivation can be adjusted by variation in the inhibitor concentration and/or exposure time in vivo selectivity of CBE and CP in cells and animal models. We envisioned that besides the traditional enzymatic assays employing fluorogenic substrates, activity‐based probes (ABPs) could be superior tools for this study. Unlike enzymatic substrate assays, which do not easily distinguish similar enzymatic activities such as GBA vs GBA2, ABPs would allow direct and unambiguous visualization of respective target glycosidases that are not occupied/inactivated by CBE or CP at the active site pocket. Cravatt and coworkers and van der Stelt and colleagues earlier used ABPs directed towards proteases and lipases in a competitive activity‐based protein profiling (ABPP) approach to identify in vivo target engagement of small compounds 3–6). Here, we report a detailed in vivo target engagement study for CBE and CP. Through parallel application of both the competitive ABPP method and enzymatic assay in lysates of cultured cells, zebrafish (Danio rerio) larvae, and brains of mice treated in vivo with a relevant range of concentrations of CBE or CP, we have systematically assessed their in vivo off‐targets and selectivity windows for GBA.Our aim was to systematically study the 3 and 4) 1 and CP 2 with a competitive ABPP method by which the irreversible occupancy of the catalytic nucleophile of GBA by the inhibitors during preincubation is assessed. As a validation, competition of ABP labelling by CBE and CP was compared to the loss of GBA activity measured using 3.75 mm 4‐methylumbelliferyl‐β‐glucoside as substrate 4c was quantified by SDS/PAGE and fluorescence scanning. IC50 values (concentrations of inhibitor yielding a 50% reduction of ABP 4c labelling) were determined and found to be 26.6 μm at 30 min CBE preincubation, and 2.30 μm at 180 min preincubation  and GBA2 (110 kDa) following SDS/PAGE analysis. First, we studied HEK293T cells expressing GBA2. Cells were exposed for 24 h to different concentrations of CBE (0.1 μm–10 mm) after which the residual amount of GBA, GBA2, GAA, GANAB and GUSB in cell lysates that can still be labelled with the appropriate ABPs , GBA2 (IC50 = 315 μm), GAA (IC50 = 249 μm), GANAB (IC50 = 2900 μm) and GUSB (IC50 = 857 μm). , GBA2 (IC50 =272 μm), GAA (IC50 = 309 μm), GANAB (IC50 = 1580 μm) and GUSB (IC50 = 607 μm)  for their possible inactivation by CBE. At the highest concentration of CBE (10 mm) tested, no significant loss of activity was observed for any of these additional lysosomal enzymes  and lysosomal β‐glucuronidase GUSB. For each of these enzymes, ABPs have been designed, and enzymatic activity assays with fluorogenic substrates established and GBA2 10 kDa fotive GBA 8–66 kDa m–100 mm) in the egg water for 5 days. The larvae were collected, lysed and analysed by the competitive ABPP method. Exposure to 100 mm CBE was found to reduce ABP labelling of all five glycosidases , GBA2 (IC50 = 890 μm), GAA (IC50 = 9550 μm), GANAB (IC50 = 4700 μm) and GUSB (IC50 = 6470 μm)  except for β‐galactosidase (IC50 = 11.2 mm) and α‐galactosidase (IC50 = 22.5 mm)  to CBE  Table . Thus, im) Table , Fig. 7.in vivo targets of CBE in brain of mice treated daily from day 8 to either day 25 with 37.5 mg CBE·kg−1 body weight or to day 14 with 100 mg CBE·kg−1 body weight −1 body weight  for 24 h. IC50 values of CP for blocking ABP labelling were 0.063 μm for GBA and 0.154 μm for GBA2   Fig. A. As detm) also comparably competed GBA and GBA2 labelling, but not that of GUSB, GAA and GANAB  4m ABP 4c. We therefore generated HEK293T cells overexpressing GBA3 to study the inhibitor sensitivity of the enzyme. In these cells we could measure the in vivo interaction of CBE and CP with GBA3 by ABP detection (ABP 4c)  by acid ceramidase‐mediated conversion of accumulating GlcCer in lysosomes m CBE; sixfold increase at 10 μm CP)  and in zebrafish larvae . Thus, a selective window for in vivo GBA inactivation by CBE exists   Fig. . ImportaOur investigation also revealed that CP is a more potent GBA inactivator. However, CP inhibits GBA2 on a par with GBA in both cultured cells and zebrafish larvae Table , and the2 (fibroblasts) or 7% CO2 (HEK293T). Zebrafish (Danio rerio) were handled and maintained according to standard protocols (zfin.org). Adult zebrafish were housed at a density of 40 per tank, with a cycle of 14 h of light and 10 h of darkness. Adults, embryos and larvae were kept at a constant temperature of 28.5 °C. Embryos and larvae were raised in egg water . Synchronized wild‐type ABTL zebrafish embryos were acquired after mating of single male and female couples (both > 3 months old). Frozen brain samples from wild‐type and CBE‐treated mice were obtained from a previous study Cyclophellitol (CP) and the ABPs were synthesized as described earlier  CO2 HEK23T. Zebram citric acid—Na2HPO4, 0.1% (w/v) bovine serum albumin) for a period of 30 min to 2 h. After stopping the substrate reaction with 200 μL 1M NaOH‐glycine (pH 10.3), 4MU‐emitted fluorescence was measured with a fluorimeter LS55  using λEX 366 nm and λEM 445 nm prism 7.0  by the [inhibitor] vs response—various slope (four parameters) method to obtain IC50 values. The substrate mixtures used for each enzyme are listed as follows: GBA, 3.75 mm 4MU‐β‐D‐glucopyranoside  at pH 5.2, supplemented with 0.2% (w/v) sodium taurocholate and 0.1% (v/v) Triton X‐100, and 25 nm N‐(5‐adamantane‐1‐yl‐methoxy‐pentyl)‐deoxynojirimycin (AMP‐DNM), a GBA2‐specific inhibitor m 4MU‐β‐D‐glucopyranoside at pH 5.8, with pre‐incubation with 1 μm ABP 3a for 30 min to specifically inhibit GBA activity; α‐glucosidases, 3 mm 4MU‐α‐D‐glucopyranoside at pH 4.0 (GAA) or at 7.0 (GANAB), GUSB, 2 mm 4MU‐β‐D‐glucuronide at pH 5.0; α‐galactosidases, 2 mm 4MU‐α‐D‐galactopyranoside at pH 4.6; β‐galactosidases, 1 mm 4MU‐β‐D‐galactopyranoside at pH 4.3 with 0.2 M NaCl; α‐mannosidases, 10 mm 4MU‐α‐D‐mannopyranoside at pH 4.0; β‐mannosidases, 2 mm 4MU‐β‐D‐mannopyranoside (Glycosynth) at pH 4.2; β‐hexosaminidase HexA, 5 mm 4MU‐β‐D‐6‐sulpho‐2‐acetamido‐2‐deoxy‐glucopyranoside at pH 4.4; β‐hexosaminidases HexA/B, 5 mm 4MU‐β‐N‐acetyl‐glucosaminide at pH 4.5; α‐N‐acetyl‐galactosaminidase, 1 mm 4MU‐α‐N‐acetyl‐galactosaminide at pH 4.5; α‐L‐fucosidase, 1 mm 4MU‐α‐L‐fucopyranoside at pH 5.0, α‐L‐iduronidase, 2 mm 4MU‐α‐L‐iduronide (Glycosynth) at pH 4.0; GBA3, 3.75 mm 4MU‐β‐D‐glucopyranoside at pH 6.0.All assays were performed in 96‐well plates at 37 °C for human, zebrafish and mice material. Samples were diluted with McIlvaine buffer to a final volume of 25 μL, at pH appropriate for each enzyme. Assays were performed by incubating the samples with 100 μL 4MU‐ (4‐methylumbelliferyl‐) substrates diluted in McIlvaine buffer  Triton‐100, 0.2% (w/v) sodium taurocholate), or labelled together with GBA2 using 200 nm β‐aziridine ABP 4c at pH 5.5. GBA2 was labelled with 200 nm β‐aziridine ABP 4a, 4b or 4c. The α‐glucosidases GAA and GANAB were first preincubated with 200 nm ABP 4a for 30 min (pH 4.0 for GAA and pH 7.0 for GANAB), followed by labelling with 500 nm ABP 6a or 6c at pH 4.0 or 7.0. The β‐glucuronidase GUSB was preincubated with 200 nm ABP 4a for 30 min, followed by labelling with 200 nm β‐aziridine ABP 5c. After ABP incubation, proteins were denatured by boiling the samples with 5× Laemmli buffer (50% (v/v) 1 M Tris‐HCl, pH 6.8, 50% (v/v) 100% glycerol, 10% (w/v) DTT, 10% (w/v) SDS, 0.01% (w/v) bromophenol blue) for 5 min at 98 °C, and separated by electrophoresis on 7.5% or 10% (w/v) SDS/PAGE gels running continuously at 90 V and curve‐fitted using prism 7.0 (GraphPad Software). After fluorescence scanning, SDS/PAGE gels were stained for total protein with Coomassie G250 and scanned on a ChemiDoc MP imager .Residual active, not irreversibly inhibited glycosidases were labelled with excess fluorescent ABPs in the optimum McIlvaine buffer, if not otherwise stated (see above). ABP labelling was performed at 37 °C for 30 min for all materials, in a total sample volume of 20–40 μL and 0.5–1% DMSO concentration. GBA was labelled with 200 ny at 90 V, 37, 38.50 measurements using enzymatic assay, 3.16 ng (53 fmole) of rGBA was prepared in 12.5 μL McIlvaine buffer  supplemented with 0.1% (v/v) Triton X‐100, and 0.2% (w/v) sodium taurocholate, and incubated with 12.5 μL of inhibitors (CBE or CP) diluted in McIlvaine buffer at 37 °C for various time periods. Residual activity of rGBA was measured as described in previous section. For assessing the occupancy of active site pocket by the inhibitors using ABP labelling, the same amount of rGBA was prepared in 10 μL of the same McIlvaine buffer, incubated firstly with 2.5 μL inhibitor dilutions prepared in McIlvaine buffer at 37 °C for various time periods, then with 2.5 μL ABP dilutions prepared in McIlvaine buffer; detection of ABP‐labelled rGBA follows the procedures described in the previous section.For ICm) or CP (0.001–10 μm) for 24 h at 37 °C. In addition, confluent human fibroblasts were similarly treated in 15‐cm dishes for 2, 24 and 72 h. For lysis, cells were washed three times with PBS, subsequently lysed by scraping in potassium phosphate buffer , aliquoted and stored at −80 °C. After determination of the protein concentration, lysates containing equal protein amount  were adjusted to 12 μL with potassium phosphate buffer and subjected to residual activity measurements using enzymatic assay  or ABP detection . For in vivo ABP labelling, HEK293T cells expressing GBA2 were incubated with culture medium containing CBE (0.015–15 000 μm) or CP (0.001–10 μm) for 24 h. Medium was removed and cells were incubated for 4 h with culture medium containing a mixture of 200 nm ABP 4b, 1 μm ABP 5c and 500 nm ABP 6c, or with DMSO only (negative control). Lysates were prepared and measured for protein concentration as described above, and samples containing 60 μg total protein  were subjected to ABP detection .Confluent HEK293T stably expressing human GBA2 were cultured in 12‐well plates in triplicates with(out) CBE , or CP (0.001–100 μm) for 120 h at 28.5 °C. Per condition, n = 48 embryos were used. At 120 h (5 dpf), larvae were collected, rinsed three times with egg water, fully aspirated, snap‐frozen in liquid nitrogen and stored at −80 °C until homogenization in 200 μL 25 mm potassium phosphate buffer per 48 individual. Lysis was conducted by sonication with a Polytron PT 1300D sonicator  on ice at 20% power for 3 s, and repeated three times. Samples containing 20–45 μg total protein were subjected to ABP detection or enzymatic assay.Adult zebrafish were not sacrificed for this study; all experiments were performed on embryos/larvae before the free‐feeding stage  and did not fall under animal experimentation law according to the EU Animal Protection Directive 2010/63/EU. For −1 body weight, or PBS, from day 8 until day of sacrifice  as previously described m potassium phosphate buffer (4× volume/wet tissue weight) with 1.0 mm glass beads using a Fastprep‐24 instrument  set at 6 m·s−1 for 20 s, repeated three times, while chilling samples on ice for 2 min between separate runs. Crude lysates were isolated from the glass beads by pipetting into sterile Safe‐Lock Eppendorf tubes. Homogenates were measured for protein concentration, aliquoted and snap‐frozen in liquid nitrogen. Samples containing 50 μg total protein were subjected to ABP detection or enzymatic assay. Two‐tailed unpaired t‐test was performed in prism 7.0 software (GraphPad Software) to derive statistical significance, where P < 0.05 was considered significant.Brain hemispheres were obtained from mice injected daily with CBE at either 37.5 mg or 100 mg·kgm ABP 4c at pH 6.0. For enzymatic assay, lysates were separated from GBA2 and GBA by centrifugation (16 000 g for 10 min at 4 °C) and incubation of the resulting supernatant with 20 μL concanavalin A sepharose beads (Sigma‐Aldrich) in 200 μL of binding buffer  for 1 h at 4 °C on a rotor. Next, beads were removed from the supernatant by centrifugation. Five microlitres of the supernatant was added with 20 μL McIlvaine buffer (pH 6.0), and subjected to enzymatic assays for GBA3 activity. Measured activity was normalized with the corresponding protein concentration of each sample, and data were processed as earlier described.HEK293T cells expressing human GBA3 were cultured, treated (triplicate sets) and lysed in an identical setup as described with GBA2‐expressing HEK293T cells. Lysates (12 μg protein) were subjected to ABP detection using 200 nm) or CP (0.01–10 μm)) for 112 h at 28 °C. Thereafter, zebrafish larvae were washed three times with egg water, and collected in clean screw‐cap Eppendorf tubes (three tubes of three larvae per inhibitor concentration). Lipids were extracted and measured according to methods described previously 13C‐GlcSph −1 in methanol (MeOH), 480 μL MeOH and 250 μL CHCl3 were added to the sample, stirred, incubated for 30 min at RT, sonicated (5 × 1 min in sonication water bath) and centrifuged for 10 min at 15 700 g. Supernatant was collected in a clean tube, 250 μL CHCl3 and 450 μL 100 mm formate buffer (pH 3.2) were added. The sample was stirred and centrifuged, the upper phase was transferred to a clean tube. The lower phase was extracted with 500 μL MeOH and 500 μL formate buffer. The upper phases were pooled and taken to dryness in a vacuum concentrator at 45 °C. The residue was extracted with 700 μL butanol and 700 μL water, stirred and centrifuged. The upper phase (butanol phase) was dried and the residue was dissolved in 100 μL MeOH. Ten microlitres of this sample was injected to the LC‐MS for lipid measurement. Two‐tailed unpaired t‐test was performed in Prism 7.0 software (GraphPad Software) to derive statistical significance, where P < 0.05 was considered significant.Zebrafish embryos at 8 h postfertilization were seeded in 12‐well plates  and treated with CBE (10–1000 μThere are no conflicts of interest declared.CLK, WWK and IZ performed the experiments; LTL, AV, AHF, AHM and HPS provided essential materials. CLK, WWK, and MM analysed the data. WWK and MM helped to write the manuscript. CLK, JMFGA and MA wrote the manuscript. AHF, HSO and MA revised the manuscript. HSO and JMFGA conceived and designed the study.


III ion is nine-coordinated in a distorted tricapped trigonal–prismatic geometry by three oxygen atoms and three nitrogen atoms from three benzhydrazide (bzz) ligands, one oxygen atom from the isophthalate (itp2−) ligand, and two oxygen atoms from coordinated water molecules. The crystal structure features extensive hydrogen bonding as well as C—H⋯π and π–π interactions.The first benzohydrazide complex of a lanthanide is reported. The Sm 8H4O4)(C7H8N2O)3(H2O)2]NO3, systematic name diaquatrissamarium(III) nitrate, the SmIII ion is nine-coordinated in a distorted tricapped trigonal–prismatic geometry by three oxygen atoms and three nitrogen atoms from three benzhydrazide (bzz) ligands, one oxygen atom from the isophthalate (itp2−) ligand, and two oxygen atoms from coordinated water molecules. The nitrate group is disordered over two sets of sites with occupancy factors of 0.310 (17) and 0.690 (17). In the crystal, adjacent molecules are linked into chains via pairs of O—H⋯O and N—H⋯O hydrogen bonds between the carboxylate acceptor and the coordinated water and amine NH2 donors. Molecules are further stacked by π–π interactions involving the benzene ring of the itp2− ligands, forming double chains that extend in the b-axis direction. These double chains are further linked into a three-dimensional supramolecular network via hydrogen bonds  between the complex molecule and the nitrate groups along with C—H⋯π and π–π interactions involving the benzene rings of the bzz and itp2− ligands.The first benzohydrazide complex of a lanthanide is reported. In the title compound, [Sm(C Distinct from transition metal centers, lanthanide ions often demonstrate high and variable coordination numbers as well as diverse coordination geometries, which can lead to versatile structures and topologies  ligands, one completely deprotonated isophthalate (itp2−) ligand, two coordinated water molecules, and one disordered NO3− ion. The hydrazide group of the bzz ligand adopts a bidentate μ2-η1:η1 chelating coordination mode, whereas the carboxylate groups of the fully deprotonated itp2− ligand display a μ1-η1:η0 monodentate coordination fashion. The SmIII ion is nine-coordinated by three oxygen atoms  and three nitrogen atoms  of three different bzz ligands, one oxygen atom (O4) from the completely deprotonated itp2− ligand, and other two oxygen atoms  from the coordinated water molecules. The central metal SmIII atom can be described as having a distorted tricapped trigonal–prismatic geometry, Fig. 2III complexes  Å and a dihedral angle = 0.0 (2)°  , and the bzz and itp2− ligands , are also observed, which help further to stabilize the crystal structure.As can be seen in Fig. 3s Table 1 involvinet al., 2016et al., 1971et al., 2003et al., 2011et al., 2009A search of the Cambridge Structural Database 3·6H2O , bzz , H2itp , and H2O (4 ml) was sealed in a 15 ml Teflon-lined steel autoclave and heated at 373 K for 24 h. The mixture was cooled to room temperature and light-yellow block-shaped crystals of the title compound were obtained in 79% yield . Analysis calculated (%) for C29H32N7O12Sm (1376.80): C 42.43; H 3.93; N 11.94%. Found: C 42.46; H 3.96; N 11.90%.A mixture of Sm(NOUiso(H) = 1.2Ueq(C). The H atoms bonded to O and N atoms were located in a difference-Fourier map, but were refined with distance restraints of O—H = 0.84 ± 0.01 Å and N—H = 0.88 ± 0.01 Å, and with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N). The nitrate group is disordered over two sets of sites, with occupancy factors of 0.310 (17) and 0.690 (17).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018015360/pj2059sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018015360/pj2059Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018015360/pj2059Isup3.cdxSupporting information file. DOI: 1876239CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S,3S)-2-amino-3-methylpentanoic acid, l-isoleucine C6H13NO2, crystallizes in the monoclinic space group P21 with four independent molecules in the asymmetric unit. In the crystal, N–H⋯O hydrogen bonds link two pairs of independent molecules and their symmetry-related counterparts to form two types of layers stacked in an anti-parallel manner parallel to (001). The hydrophobic aliphatic isopropyl groups protrude from these layers.A new polymorph of -2-amino-3-methylpentanoic acid, l-isoleucine C6H13NO2, crystallizes in the monoclinic space group P21 with four independent molecules in the asymmetric unit. The molecules are zwitterions. In the crystal, N—H⋯O hydrogen bonds link two pairs of independent molecules and their symmetry-related counterparts to form two types of layers stacked in an anti-parallel manner parallel to (001). The hydrophobic aliphatic isopropyl groups protrude from these layers.A new polymorph of (2 S,3S)-2-Amino-3-methylpentanoic acid, known as l-isoleucine (l-Ile), is one of the 20 amino acids common in animal proteins and required for normal functioning in humans. l-Ile is classified as a hydrophobic amino acid and is one of the two common amino acids that has a chiral side chain. l-Ile is essential for human muscle tissue recovery after exercise, along with Valine and Leucine.°, N1—C2—C3—C4 = −155.4 (3)° and N3—C14—C15—C18 = 78.1 (4)°, N3—C14—C15—C16 = −155.8 (3)°. The other two molecules are of conformer type II with the torsion angles N2—C8—C9—C12 = 178.6 (4)°, N2—C8—C9—C10 = −56.9 (5)° and N4—C20—C21—C24 = 179.1 (4)°, N4—C20—C21—C22 = −56.8 (5)°. Furthermore, there is a minor conformational variance between all the four independent molecules, as illustrated by the torsion angles of the iso-propyl side chains: C6—C3—C4—C5 = −56.6 (5)°, C12—C9—C10—C11 = −51.6 (6)°, C18—C15—C16—C17 = −58.9 (5)° and C24—C21—C22—C23 = −53.2 (6)°.The existence of another chiral center in the side chain allows for conformational differences. Each l-Ile is embedded inside the layers while the side chains are oriented away, creating a hydrophobic surface. However, this hydrogen-bonding network has directionality along the polar b axis and specifically parallel to (001)  was dissolved in water  by heating to 353 K, with constant stirring until total dissolution. The hot solution was then filtered through cotton wool into glass crystallization dishes, which were covered with filter paper in order to allow slow evaporation, placed in a heating bath. Colorless crystal chunks, suitable for X-ray crystallographic analysis were obtained. The absolute configuration of the title compound is already known.Single crystals of Uiso(H) = 1.2Ueq(C) or 1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018006126/lh5872sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018006126/lh5872Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018006126/lh5872Isup3.cmlSupporting information file. DOI: 1838774CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: