Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation. •The MDGA1 extracellular region has an unusual triangular multi-domain arrangement•The NL1-MDGA1 complex structure reveals how MDGA proteins block neurexin binding•MDGA1 and MDGA2 bind all NL isoforms, a process fine-tuned by alternative splicing•MDGA1 and MDGA2 suppress NL synaptogenic activity in a concentration-dependent manner Elegheert et al. present the crystal structure of the autism-linked post-synaptic protein MDGA in complex with the synapse organizer neuroligin, providing a structural and mechanistic basis for potentially brain-wide modulation of synaptic neuroligin-neurexin signaling by MDGA proteins. A receptors through direct interactions or using DLG (Discs large) family or gephyrin and collybistin accessory proteins, respectively  and B (SSB). Mammalian NRXs show even greater diversity: over a thousand variants are generated from three genes, two promoters (α and β), and six sites of alternative splicing (SS1–6) . The extBesides NLs, the various NRXs bind a multitude of postsynaptic protein families to organize synapses: leucine-rich repeat transmembrane proteins (LRRTMs), calsyntenin 3, dystroglycan, latrophilin 1, cerebellins (reviewed in 1-6), a fibronectin type III-like (FnIII7) domain, and a memprin, A5, mu (MAM8) domain.In contrast to all these positive effectors and modulators, the discovery of the Ig superfamily (IgSF) MDGA  proteins as negative modulators of NL is remarkable. MDGA1 was found to block the interaction of NL2 with NRX and suppress inhibitory synapse development in cultured neurons , while MMDGA1 are linked to schizophrenia  and schizophrenia . Similarophrenia , and MDGs of ASD . Single-d to ASD . Mdga2 hignaling . Howeverignaling , raisingDespite the recent focus on mapping the complex molecular landscape of NL-NRX signaling modulators, a structural and mechanistic understanding of these processes is still lacking. In this study, we present the crystal structure of the near-complete MDGA1 extracellular domain and that of its prototypical complex with NL1, providing detailed insight into the structural basis of the modulation of NL-NRX signaling by MDGA proteins. We show that human MDGA1 and MDGA2 have the ability to interact with human NL1–5, thereby extending the previously proposed restricted, binary NL-MDGA interaction code . FurtherECTO; Ig1-Mam8; Gln19-Lys919; 79.5% sequence identity and 88.4% sequence similarity with human MDGA1ECTO; ECTO was treated with endoglycosidase F1 (Endo F1) prior to crystallization, leaving a single N-linked N-acetylglucosamine monosaccharide on glycosylated Asn residues after enzymatic cleavage. Seven domains (Ig1-6 to FnIII7) could be unequivocally resolved in the electron density maps; however, the C-terminal MAM8 domain was not visible and most likely highly mobile and accommodated in the solvent channels of the crystal. The cMDGA1ECTO Ig1-6-FnIII7 domains form a surprisingly compact, folded structure that is ∼120 Å wide, ∼110 Å high, and ∼50 Å deep, fitting comfortably within the typical height of the synaptic cleft (∼20–25 nm). Its approximately triangular shape, unique among the cell-surface receptors crystallized to date, is a consequence of sharp-angled Ig2-Ig3, Ig4-Ig5, and Ig6-FnIII7 inter-domain linkers that are stabilized by numerous inter-domain contacts.As a first step toward solving the structure of an NL-MDGA complex, we targeted the full-length apo MDGA1 extracellular domain for crystallization. Following an extensive screen of constructs from various species, we obtained diffraction-quality crystals and solved the structure of the complete chicken MDGA1 extracellular region (cMDGA12-Ig3 domain contacts (341 Å2 buried surface area [BSA]) are formed between (1) the Ig2 β strands βA and βG and (2) the loop structure connecting Ig2 and Ig3, and also Ig3 loops BC and FG. The Ig4-Ig5 domain contacts (598 Å2 BSA) are formed between (1) the Ig4 β strand βA and loop AB and (2) Ig5 loops BC and FG. The Ig6-FnIII7 domain contacts (396 Å2 BSA) are formed between (1) the Ig6 β strand βA′ and loops A′B and EF and (2) the loop connecting Ig6 and FnIII7 and FnIII7 loops BC and FG. Finally, the Ig1-FnIII7 domain contacts (395 Å2 BSA) close the cMDGA1ECTO triangle and are formed between (1) the Ig1 N-terminal stretch (Gln19-Tyr22) and loop BC and (2) FnIII7 loops AA′ and C′E, and β strands βA and βB ; their combined BSA is 2,666 Å2, suggesting a stable association. Interestingly, this arrangement is compatible with both a potential cis- or trans-homophilic interaction and might indicate formation of an adhesive or self-inhibitory complex  at a concentration of 30 μM. The scattering data were unambiguously incompatible with a dimeric MDGA1 molecule but were instead accurately (χ2 = 1.17) modeled as a limited ensemble of monomeric conformers with pronounced flexibility at the FnIII7-Mam8 domain linkage  for binding to wild-type cMDGA1ECTO. Both cMDGA1ECTO variants failed to interact with wild-type cMDGA1ECTO up to a concentration of 100 μM  between apo and NL1-bound cMDGA1ECTO structures is only 1.5 Å over 647 Cα atoms, underlining the stability and importance of this unusual multi-domain architecture. The NL1 and MDGA1 C termini point in the same direction and thus confirm an interaction in cis, situated on the postsynaptic membrane , Q0WYX8 (chicken MDGA1), and Q8N2Q7 (human NL1). Annotation of secondary structural elements follows the acetylcholinesterase (AChE) nomenclature  is formed between residues from (1) MDGA1Ig1 β strands C, F, and G and loop CE and (2) NL1 loops Leu289-Gln307, Ile377-Asp385 (part of “loop L1”), Gln392-Tyr398, and Phe496-Pro499 and helices α2 and α4  contacts and form putative hydrogen bonds with multiple MDGA1Ig1 residues. AspNL1384 and GluNL1394 form putative salt bridges with ArgMDGA1105 and ArgMDGA1123  B. HisNL1MDGA1123 A.2 BSA) is formed between residues from (2) MDGA1Ig2 β strands A, B, D, and E and NL1 α helices α2 and α3; (2) MDGA1Ig2 loop ABIg2 and NL1 loops Ala110-Pro132 (“Cys loop”) and Asp361-Asp385 (“loop L1”); and (3) peripheral interactions contributed by MDGA1Ig3 to NL1 α helix α2 and loop Val417-Ser424   could not be resolved in the complex electron density map  B. The Phsity map A.The NL1 “Cys loop” (part of loop Ala110-Pro132) and “loop L1” (part of loop Asp361-Asp385) occlude the “gorge” that, in AChE, leads to the enzyme active site. Interestingly, these loop structures form an integral part of the NL-MDGA interface. In this sense, MDGA resembles the snake toxin fasciculin (Fas) for binding to AChE . There aV2.2 voltage-gated calcium channel. Furthermore, the LRE motif is present in the majority of mammalian AChEs, and besides in NL, it is also observed in the cholinesterase-like adhesion molecules neurotactin and glutactin (DEIg2 (The function of the NL Leu449-Arg450-Glu451 (LRE) adhesion motif, conserved in all NLs and located in the α3 helix B and S3Clutactin . Both Arn  on the cMDGA1ructures C and S4D3B3Q; 2.4 Å; MDGA1123 mimics Argβ-NRX1232 for binding to AspNL1384. ArgMDGA1123 and ArgMDGA1105 engage GluNL1394 in ionic interactions, whereas in NL1-β-NRX1, the latter residue contacts Thrβ-NRX1235 and is part of the hexadentate coordination shell of the obligate interface calcium atom. AspNL1294 forms a hydrogen bond with TyrMDGA1107, whereas it forms a bifurcated ionic interaction with Argβ-NRX1109 in NL1-β-NRX1. Finally, NL1 residues Gly393, Phe395, Phe496, and Asn397 are contacted by MDGA1Ig1 loop CE, preventing their network of hydrogen-bonded interactions with β-NRX1 residues ) for binding to NL1ECTO. Titration of β-NRX1LNS6(–4) into NL1ECTO alone revealed a strong exothermic interaction and a KD of ∼390 nM. Application of an equimolar amount of MDGA1ECTO to NL1ECTO in the titration cell did not fully block the NL1ECTO-β-NRX1LNS6(–4) interaction, but decreased its apparent KD  ∼12-fold to 4.76 μM. Application of a 2.5-fold molar excess of MDGA1ECTO over NL1ECTO was required to fully block binding of β-NRX1LNS6(–4) to NL1ECTO  assay to investigate whether MDGA1 NL1ECTO C. These ECTO and β-NRX1LNS6 with and without SS4 (β-NRX1LNS6(±4)). The reference interaction of NL1ECTO with β-NRX1LNS6(–4) showed an approximately 2-fold higher equilibrium dissociation constant (KD) than the one derived from ITC  to low micromolar (μM) range, similar to NL-β-NRX1(±4) . Interaction affinities of MDGA2ECTO and β-NRX1LNS6(–4) for NL1–2ECTO are nearly identical. Interestingly, both MDGA1 and MDGA2 interacted ∼10- to ∼20-fold weaker with NL3, NL4, and NL5 (KD of ∼15–25 μM). Whereas NL3ECTO also binds β-NRX1LNS6(–4) with low affinity (KD of ∼8.5 μM), NL4ECTO and NL5ECTO still bind β-NRX1LNS6(–4) relatively strongly (KD of ∼2.5–3 μM), meaning that for NL4 and NL5, a larger discrepancy between binding strengths of β-NRX1(–4) and MDGA1-2 exists  A. Accord2 exists A.Taken together, these experiments show (1) that MDGA1 and MDGA2 have the ability to interact with NLs that localize to excitatory glutamatergic (NL1 and NL3) , inhibit1-3 (MDGA1ΔIg1–3)  rat brain synaptosome extracts, using affinity chromatography coupled with mass spectrometry and bioinformatics analysis . For ext1ΔIg1–3) , demonst1ΔIg1–3) B. In the1ΔIg1–3)  and NL5 1ΔIg1–3) A.FL) and full-length N-terminally HA-tagged MDGA1-2 (HA-MDGA1-2FL) variants were co-cultured with rat hippocampal neurons  N-terminally myc-tagged NL1–4 , all three proteins failed to interact with NL1(–A–B)ECTO up to a concentration of 25 μM (Given that the interactions of thrombospondin 1 (TSP1) , hevin , and theof 25 μM B and S7CΔSite I: His291Ala, Tyr292Ala, Asp384Ala, and Glu394Ala) and five into the Site II interface   A. We optConsistent with both NL-MDGA and NL-β-NRX1 complex structures, we found using SPR that the ΔSite II mutant blocked MDGA1 binding but maintained binding of β-NRX1, whereas the ΔSite I and combined ΔSite I+II mutants fully abolished both β-NRX1 and MDGA1 interactions B and S8AΔSite I and NL1ΔSite I+II mutations, but not the NL1ΔSite II mutations, prevented NL-NRX-induced synapse formation  leads to a number of ASD-linked phenotypes in mice . In thisECTO-cMDGA1ECTO complex crystal structure shows that NL1 Arg450, which is equivalent to NL3 Arg451 and part of the NL1 Leu449-Arg450-Glu451 (LRE) motif (ECTO and NL3(–A)ECTO, respectively. We observed diminished secretion for the mutants as compared to wild-type proteins (Our hNL1E) motif B and S3CE) motif A. We intproteins B, consisproteins . Using Sproteins C and S8Bproteins B.Figure Using the co-culture assay, we tested the impact of the R451C mutation on the recruitment of synaptic terminals by full-length NL3. Importantly, although impaired relative to wild-type NL3, the R451C mutant can traffic to the surface of transfected COS-7 cells C and rat3VKF; Alternative splicing leads to insertion of SSA and SSB onto the NL cholinesterase scaffold. SSB is restricted to NL1, whereas distinct SSA sequences are present in NL1, NL2, and NL3. In NL1 and NL3, the two possible SSA sequences (A1 and A2) can also occur in tandem (denoted as A1A2) B. Wherea5 and Ig6 domains  pair ) at 2.55 Å . The ninindrance B. Indeedindrance C.Using the co-culture assay, we tested the effect of the presence of SSB on the ability of MDGA1-2 to block recruitment of synaptic terminals by full-length NL1(–B) and NL1(+B). Co-expression of MDGA1-2 at low, medium, and high plasmid ratios , and NL . The complexity of NL-NRX signaling is compounded even further by the existence of postsynaptic omplexes  and by tomplexes .The capacity of NLs to form heterodimers  will difThe direct interaction affinities with NL1 and NL2 do not seem to account for selectivity of MDGAs to suppress excitatory or inhibitory synapses. Consistent with the role of MDGA2 to suppress excitatory synapses in vivo , MDGA2, In the rat and mouse brain, MDGA1 and MDGA2 are widely expressed by neuronal populations in both the central and peripheral nervous systems. These include neurons of the basilar pons, inferior olivary nucleus, cerebellum, cerebral cortex, olfactory bulb, spinal cord, dorsal root and trigeminal ganglia, and hippocampus . There ahttps://gene.sfari.org). Interestingly, two mutations in the MDGA interaction-selective Site II of NL3 have been reported in patients with ASD: Arg451Cys  and Gly426Ser     A and 7B. Ala425)  and incr Ala425) , despite Ala425) . Nonethe Ala425) C and rat Ala425) D. The la Ala425) . This al Ala425) . Importa Ala425) D, suggesAn E/I imbalance surpassing the capacity of neuronal populations and circuits to regulate synaptic homeostasis is a proposed hallmark of ASD . Disruptradu@mrc-lmb.cam.ac.uk).Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, A. Radu Aricescu , human MDGA1 , human MDGA2 , human neuroligin-1 , human neuroligin-2 , human neuroligin-3 , human neuroligin-4 (NL4 or NL4(X); GenBank: NM_020742.3; Gln42-Glu602), human neuroligin-5 (NL5 or NL4(Y); GenBank: NM_014893.4; Gln42-Glu602), human β-neurexin-1 , human thrombospondin-1 , human hevin , mouse α-Neurexin-1 .List of cDNAs and construct boundaries for secreted protein production: chicken MAM domain-containing glycosylphosphatidylinositol anchor protein (MDGA) 1  days post-transfection, the conditioned Dulbecco’s Modified Eagle Medium (DMEM) medium was collected and buffer-exchanged using a QuixStand benchtop diafiltration system  and proteins were purified by immobilized metal-affinity chromatography (IMAC) using pre-packed Nickel Sepharose columns . Proteins were concentrated and further purified by size-exclusion chromatography  in 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.50, 150 mM sodium chloride and 3 mM calcium chloride (HBS-C).These cDNAs were fused C-terminally with a hexa-histidine (His6) tag or Avitag3, and were cloned into the pHLsec vector . For lar−/−  (for cryThe rat GluN1a-GluN2B heterotetrameric NMDA receptor (NMDAR) was expressed and purified as previously described , with thA multiple-step overlap-extension PCR  was used for site-directed mutagenesis, construction of chimeric protein constructs and introduction or deletion of splice inserts ; the resQ8N2Q7; between Asp164 and Asp182);The following internal primer pair was used for the introduction of human NL1 spliced sequence “A1” (VKRISKECARKPGKKICRKG) into human NL1(–A ± B)  into human NL1(–A ± B) (between Asp164 and Asp182);FP: 5′-GAAACAGACAGATGATTTAGGTGATAATGACGGTGCTGAAGATGAAGATATTCGGGACAGTGGGGGTCCCAAACCAG-3′RP: 5′-CTTGCCGGGCTTTCTGGCACATTCCTTGGATATTCTTTTTACATCCTCAGTCGGGACATATATATTTAAATATAG-3′The following internal primer pair was used for the introduction of human NL1 spliced sequence “A2” (GPLTKKQTDDLGDNDGAEDE) into human NL1(+A1 ± B) (after spliced sequence A1);FP: 5′-GAAACAGACAGATGATTTAGGTGATAATGACGGTGCTGAAGATGAAGATATTCGGGACAGTGGGGGTCCCAAACCAG-3′RP: 5′-CATTATCACCTAAATCATCTGTCTGTTTCTTTGTAAGGGGACCTCCTTTTCTACATATTTTCTTGCCGGGCTTTC-3′The following internal primer pair was used for the introduction of human NL1 spliced sequence “B” (NRWSNSTKG) into human NL1(±A–B) (between Gly295 and Leu305);FP: 5′-GTAACCGTTGGAGCAATTCAACCAAAGGACTTTTTCAACGAGCAATAGCTCAAAG-3′RP: 5′-GTCCTTTGGTTGAATTGCTCCAACGGTTACCTTCAGAATAATGGGATAAAGTC-3′The following internal primer pairs were used for constructing the human NL1 ΔSite I mutant (H291A-Y292A-D384A-E394A);HYSEGL to LTLSAASEGL);H291A Y292A (LTLSFP: 5′-GTCAACCTGCTGACTTTATCCGCTGCTTCTGAAGGTCTTTTTCAACGAG-3′RP: 5′-CTCGTTGAAAAAGACCTTCAGAAGCAGCGGATAAAGTCAGCAGGTTGAC-3′DDPQI to VIPADPQI);D384A (VIPFP: 5′-GGTGATGTAATACCAGCCGACCCCCAGATATTG-3′RP: 5′-CAATATCTGGGGGTCGGCTGGTATTACATCACC-3′EFLNY to MEQGAFLNY);E394A ;DFAVSNFVDN to ASDFAAAVAAFVDN);D429A F430A S433A N434A (ASDFFP: 5′-GCCGCTGCTGTTGCAGCTTTTGTTGATAATTTATATGGATATCCTGAAGGCAAAGATG-3′RP: 5′-AGCTGCAACAGCAGCGGCAAAATCACTAGCTGATATACCATCATCGCTATCTAC-3′RETIK to KDVLAETIK);R450A (KDVLFP: 5′-GAAGGCAAAGATGTTTTGGCAGAAACCATTAAGTTCATG-3′RP: 5′-CATGAACTTAATGGTTTCTGCCAAAACATCTTTGCCTTC-3′RETI to DVLCETI);The following internal primer pair was used for introducing the R450C mutation into human NL1(–A–B) (DVLFP: 5′-GGCAAAGATGTTTTGTGCGAAACCATTAAGTTC-3′RP: 5′-GAACTTAATGGTTTCGCACAAAACATCTTTGCC-3′NSTKG to NRWSQSTKG);The following internal primer pair was used for introducing the Asn300Gln (N300Q) mutation into human NL1 spliced sequence “B” ;The following internal primer pair was used for the introduction of human NL2 spliced sequence “A” (GPLTKKRDEATLNPPDT) into human NL2(–A) ;The following internal primer pair was used for the introduction of human NL3 spliced sequence “A1” (VKRISKECARKPNKKICRKG) into human NL3(–A)  into human NL3(–A) (between Asp152 and Asp193);FP: 5′-GAAACAGGGCGAGGACTTAGCGGATAATGACGGGGATGAAGATGAAGACATCCGGGACAGTGGTGCTAAACCCGTC −3′RP: 5′-CCGCTAAGTCCTCGCCCTGTTTCTTAGCGCCGGATCCATCCTCCGTCGGCACATAGACGTTC-3′The following internal primer pair was used for the introduction of human NL3 spliced sequence “A2” (GSGAKKQGEDLADNDGDEDE) into human NL3(+A1) (after spliced sequence A1);FP: 5′-GAAACAGGGCGAGGACTTAGCGGATAATGACGGGGATGAAGATGAAGACATCCGGGACAGTGGTGCTAAACCCGTC-3′RP: 5′-CATTATCCGCTAAGTCCTCGCCCTGTTTCTTAGCGCCGGATCCTCCTTTCCTACAAATTTTCTTGTTGGGCTTTC-3′RETI to DTLCETI);The following internal primer pair was used for introducing the R451C mutation into human NL3(–A) ;The following internal primer pair was used for introducing the R120K mutation into chicken MDGA1 ;R156N (TVFLFP: 5′-GAGAAGACTGTCTTCCTCAATTGTACCGTCAACTCCAAC-3′RP: 5′-GTTGGAGTTGACGGTACAATTGAGGAAGACAGTCTTCTC-3′SVSRD to LRLENVSRD);S502N (LRLEFP: 5′-GGAAGCTGCGCCTGGAGAATGTCAGCCGAGACATGAG-3′RP: 5′-CTCATGTCTCGGCTGACATTCTCCAGGCGCAGCTTCC-3′RNTIQ to LAQNNTIQ);R680N ;The following internal primer pair was used for the introduction of human β-NRX1 spliced sequence #4  into human β-NRX1(–4) ;The following internal primer pair was used for introducing the C992S mutation  into humFP: 5′-CAGACTGTCAACAGTGATCCTGGACTC-3′RP: 5′-GAGTCCAGGATCACTGTTGACAGTCTG-3′Crystallization trials, using 100 nL protein solution plus 100 nL reservoir solution in sitting drop vapor diffusion format, were set up in 96-well Greiner plates using a Cartesian Technologies robot .ECTO , containing the Arg120Lys mutation, concentrated to 5.0 g/L and treated with endoglycosidase F1  for 30 min at 294K immediately prior to dispensing the crystallization drops, crystallized in 0.1M HEPES pH7.5, 4% w/v polyethylene glycol 8000. The Arg120Lys mutation was introduced into cMDGA1ECTO to bring the sequence in line with rat, mouse and human isoforms  PEG200.Crystals of cMDGA1ed stock  was usedECTO (hNL1(–A+B)ECTO; Gln46-Asp635), concentrated to 10.0 g/L, crystallized in 0.2M KSCN, 0.1M Bis-tris propane pH 8.5, 20% w/v PEG3350. Crystals were cryoprotected using reservoir solution containing 20% (v/v) PEG200.Purified glycosylated human NL1(–A+B)ECTO–cMDGA1ECTO complex, purified hNL1(–A–B)ECTO  and cMDGA1ECTO  were mixed as follows; 80 μL hNL1(–A–B)ECTO was combined with 102 μL cMDGA1ECTO (resulting in a 1:1.25 NL1:MDGA1 monomer-to-monomer molar stoichiometric ratio), 18 μL purification buffer , and 50 μL dilution buffer . The final concentration of hNL1(–A–B)ECTO thus was 0.93 g/L; that of cMDGA1ECTO was 1.76 g/L; and that of NDSB-256 was 250 mM. This preparation was treated with endoglycosidase F1  for 30 min at 294K immediately prior to dispensing the crystallization drops. The hNL1(–A–B)ECTO–cMDGA1ECTO complex crystallized in 0.1M Na.HEPES pH 7.0, 7.5% w/v PEG8000. Crystals were cryoprotected using reservoir solution containing 33% (v/v) PEG200.To crystallize the hNL1(–A–B)ECTO were collected at Diamond Light Source (DLS) beamline I03 to a nominal resolution of 3.20 Å in space group (SG) P212121. X-ray fluorescence wavelength scans were performed to experimentally determine the Selenium absorption K-edge peak. The cMDGA1ECTO structure was determined using Single Anomalous Diffraction (SAD); the heavy-atom Selenium substructure was solved using SHELXD (Diffraction data for cMDGA1g SHELXD  at 3.70 g SHELXD . AutomatECTO were collected at Diamond Light Source (DLS) beamline I24 to a nominal resolution of 2.55 Å in SG P22121. The structure was determined by molecular replacement using the program Phaser (3BIX) crystal structure as search model.Diffraction data for hNL1(–A+B)m Phaser , and usiECTO–cMDGA1ECTO complex were collected at Diamond Light Source (DLS) beamline I04-1 to a nominal resolution of 3.30 Å in SG P21212. The structure was determined by molecular replacement using the program Phaser (ECTO (in which the spliced sequence B was excised from the molecular model) and cMDGA1ECTO crystal structures we determined here as search models.Diffraction data for the hNL1(–A–B)m Phaser , employiAll data were indexed, integrated, and scaled using the automated XIA2 expert system , using tECTO, hNL1(–A+B)ECTO and the hNL1(–A–B)ECTO–cMDGA1ECTO complex was initially performed with Refmac using “jelly body” restraints  was treated with endoglycosidase F1  for 12 hr at 294K and re-purified using SEC in 10 mM HEPES pH 7.50, 150 mM NaCl. SAXS data were collected at beamline BM29 of the European Synchrotron Radiation Facility  (q) range of 0.01 Å−1 < q < 0.45 Å−1, where q = 4πsin(θ)/λ, and 2θ is the scattering angle. The X-ray wavelength was 0.9950 Å, and data were collected on a Pilatus 1M detector. cMDGA1ECTO was measured at concentrations of 1.50 and 3.36 g/L in 10 mM HEPES pH 7.50, 150mM NaCl. Data reduction and calculation of invariants was carried out using standard procedures implemented in the ATSAS (Purified cMDGA1 France)  at 293 Khe ATSAS  and ScÅthe ATSAS  suites. 8 domain was generated by homology modeling starting from the crystal structure of the N-terminal RPTPmu MAM domain    using th P28827) . This mo MODELER  “Model/R MODELER .2 = 1.17) modeled as a five-membered ensemble of monomeric conformers with pronounced flexibility at the FnIII7-Mam8 domain linkage.Coarse-grained molecular dynamics (MD) simulations were performed using the program Allosmod . Five incDNA for the immobilized proteins was cloned into the pHLsec-Avitag3 vector , resultiDue to (i) sample consumption associated with equilibrium affinity experiments of high-nanomolar to low-micromolar interactions and (ii) the limited production yield of MDGA1 and −2 proteins, we prioritized testing the full matrix of NL–MDGA isoform interactions over performing replicate experiments of only a selected number of interactions.ECTO and cMDGA1ECTOGLYCAN WEDGE (triple glycan wedge (GW) mutant; Arg680Asn-Ser502Asn-Arg156Asn) variants were immobilized at a level of 2000 RU to maximize the likelihood of detecting a potentially weak binding event. SPR running buffer composition was TBS-CT supplemented with 1.0 g/L bovine serum albumin  as passivating agent to prevent binding to the carboxymethyldextran-based SPR chips. MDGA1ECTO was prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 18 concentrations of cMDGA1ECTO prepared in a two-fold dilution series from a 100 μM stock concentration was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. No self-association binding event could be detected.cMDGA1ECTO, NL2(–A)ECTO, NL3(–A)ECTO, NL4ECTO and NL5ECTO were immobilized at a level of 500 RU. SPR running buffer composition was TBS-CTB. β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO were prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 15 concentrations of β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO, prepared in a two-fold dilution series from a 50 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of MDGA1ECTO and MDGA2ECTO, the surfaces were regenerated using consecutive 30 s injections of 10 mM Tris pH 7.4, 100 mM L-Arginine/L-Glutamate, 1M NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and data was fitted to a 1:1 Langmuir binding model in Prism 6 (Graphpad).NL1(–A–B)ECTO(–4) were immobilized at a level of 2000 RU. SPR running buffer composition was TBS-CTB. Human NL1(–A–B)ECTO was prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 14 concentrations of NL1(–A–B)ECTO, prepared in a two-fold dilution series from a 25 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of the interaction with α-NRX1ECTO(–4), the surfaces were regenerated using a 30 s injection of 10 mM Tris pH 8.0, 350 mM EDTA, 100 mM NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and the α-NRX1ECTO(–4) data was fitted to a two-state Langmuir binding model in Prism 6 (Graphpad).Human hevin, human TSP1 and mouse α-NRX1ECTO, NL1(–A–B)ECTO ΔSite I, NL1(–A–B)ECTO ΔSite II and NL1(–A–B)ECTO ΔSite I+II were immobilized at a level of 550 RU. SPR running buffer composition was TBS-CTB. β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO were prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 15 concentrations of β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO, prepared in a two-fold dilution series from a 50 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of MDGA1ECTO and MDGA2ECTO, the surfaces were regenerated using consecutive 30 s injections of 10 mM Tris pH 7.4, 100 mM L-Arginine/L-Glutamate, 1M NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and data was fitted to a 1:1 Langmuir binding model in Prism 6 (Graphpad).NL1(–A–B)ECTO and NL1(–A–B)ECTO Arg450Cys were immobilized at a level of 500 RU. NL3(–A)ECTO and NL3(–A)ECTO Arg451Cys were immobilized at a level of 1000 RU. SPR running buffer composition was TBS-CTB. β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO were prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 15 concentrations of β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO, prepared in a two-fold dilution series from a 50 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of MDGA1ECTO and MDGA2ECTO, the surfaces were regenerated using consecutive 30 s injections of 10 mM Tris pH 7.4, 100 mM L-Arginine/L-Glutamate, 1M NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and data was fitted to a 1:1 Langmuir binding model in Prism 6 (Graphpad).NL1(–A–B)ECTO, NL1(+A1–B)ECTO, NL1(+A2–B)ECTO and NL1(+A1+A2–B)ECTO were immobilized at a level of 500 RU. SPR running buffer composition was TBS-CTB. β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO were prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 15 concentrations of β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO, prepared in a two-fold dilution series from a 50 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of MDGA1ECTO and MDGA2ECTO, the surfaces were regenerated using consecutive 30 s injections of 10 mM Tris pH 7.4, 100 mM L-Arginine/L-Glutamate, 1M NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and data was fitted to a 1:1 Langmuir binding model in Prism 6 (Graphpad).NL1(–A–B)ECTO, NL2(+A)ECTO, NL3(–A)ECTO, NL3(+A1)ECTO, NL3(+A2)ECTO and NL3(+A1+A2)ECTO were immobilized at a level of 500 RU. SPR running buffer composition was TBS-CTB. β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO were prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 15 concentrations of β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO, prepared in a two-fold dilution series from a 50 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of MDGA1ECTO and MDGA2ECTO, the surfaces were regenerated using consecutive 30 s injections of 10 mM Tris pH 7.4, 100 mM L-Arginine/L-Glutamate, 1M NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and data was fitted to a 1:1 Langmuir binding model in Prism 6 (Graphpad).NL2(–A)ECTO, NL1(–A+B)ECTO, and NL1(–A+B Asn300Gln)ECTO were immobilized at a level of 500 RU. SPR running buffer composition was TBS-CTB. β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO were prepared by SEC in TBS-CT. BSA was added to the concentrated stock solutions to a final concentration of 1.0 g/L. Injection of 15 concentrations of β-NRX1LNS6(–4), β-NRX1LNS6(+4), MDGA1ECTO and MDGA2ECTO, prepared in a two-fold dilution series from a 50 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 150 s at a flow rate of 25 μL/min, followed by a 180 s dissociation phase. In the case of MDGA1ECTO and MDGA2ECTO, the surfaces were regenerated using consecutive 30 s injections of 10 mM Tris pH 7.4, 100 mM L-Arginine/L-Glutamate, 1M NaCl. Equilibrium binding analysis was performed using Scrubber 2.0 (BioLogic Software) and data was fitted to a 1:1 Langmuir binding model in Prism 6 (Graphpad).NL1(–A–B)2, 0.010% LMNG. For the interaction with NL1(–A–B)ECTO, a single-cycle kinetics (SCK) approach was adopted. Injection of 5 concentrations of NL1(–A–B)ECTO, prepared in a two-fold dilution series from a 25 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 120 s at a flow rate of 25 μL/min, followed by a 60 s intermittent dissociation phase or a final 600 s dissociation phase.SPR experiments were performed on a Biacore T200 machine  operated at a data collection frequency of 10 Hz; i.e. a temporal resolution of 0.1 s. Streptactin XT (IBA Lifesciences) was chemically coupled via amine coupling chemistry onto CM5 chips to a response unit (RU) level of 5000 RU. Then, OneStrep-tagged rat GluN1a-GluN2B heterotetrameric NMDA receptor (NMDAR) was captured to a level of 5000 RU. SPR running buffer composition was 200 mM NaCl, 20 mM HEPES pH 7.4, 10 mM Glycine, 10 mM Glutamate, 3mM CaClH°), apparent entropy (ΔS°), association constant (KA), and binding stoichiometry (N) was determined.Calorimetric measurements were carried out using samples purified by SEC in HBS-C buffer . Experiments were carried out using a VP-ITC MicroCalorimeter  at 295 K, and data were analyzed using the Origin ITC analysis software package. Titrations were always preceded by an initial injection of 3 μL and were carried out using sequential 10 μL injections with continuous stirring. The data were fitted to the “one binding site model” and apparent molar reaction enthalpy (ΔECTO was concentrated to 60 μM (6.31 g/L) in TBS-CT buffer. Samples were held in Epon sector-shaped 2-channel centerpieces (6 mm path length) and were spun at 40,000 rpm. 200 sample distribution scans were taken incrementally, spaced 4 min apart. Data were collected using 280 nm absorbance optics.Sedimentation velocity (SV) experiments were performed using a Beckman Optima XL-I analytical centrifuge operated at a run temperature of 293K. Human MDGA1SMIN = 0.0, SMAX = 20, and a resolution value of 100. A value of 0.73 mL/g was used for the partial specific volumes. A buffer density value of 1.00527 g/cm3 and buffer viscosity value of 0.01022 Poise was calculated using the Sednterp online application. Figures were prepared using the program GUSSI , were cloned into the pHLsec vector  human MDGA1 (Gln19-Arg955), and human MDGA2 (Gln21-Arg956), fused N-terminally with a HA epitope tag, and without C-terminal tags (yielding HA-MDGA1-2c vector .FL), were cloned into the pHLsec vector (FL_Arg450Cys and NL3(–A)FL_Arg451Cys used native signal sequences and were not C-terminally fused to ECFP, to avoid the potential impact of these modifications on surface trafficking of the mutants.Constructs of the full-length human NL1 , human NL2 , human NL3 , and human NL4 (NL4(X); Gln42-Val816), fused N-terminally with a Myc epitope tag and fused C-terminally with ECFP  0.5 μg myc-NL1-4FL and 1.75 μg HA-CD4, 1.75 μg HA-MDGA1FL, or 2.5 μg HA-MDGA2FL for medium ratio experiments; and (iii) 0.5 μg myc-NL1-4FL and 2.5 μg HA-CD4, 2.5 μg HA-MDGA1FL, or 3.6 μg HA-MDGA2FL for high ratio experiments. HA-MDGA1FL and HA-MDGA2FL plasmid DNA amounts were adjusted to achieve similar surface protein levels. One day post-transfection, COS-7 cells were seeded onto 14 day in vitro (DIV) hippocampal cultures. After 20-24 hr the co-cultures were fixed in 4% paraformaldehyde (PFA) and 4% sucrose in PBS (pH 7.4) for 12 min at room temperature and incubated with blocking solution  for 30 min at 310K. Surface NLs and MDGAs were labeled by incubating with anti-myc and anti-HA antibodies, respectively, for 1 hr at 310K. The cells were permeabilized with 0.2% Triton X-100 in PBS, blocked for 30 min at 310K and incubated with anti-synapsin1 and anti-tau antibodies overnight at 277K. Secondary antibodies were applied for 30 min at 310K and the coverslips were mounted onto glass slides with elvanol .Low density primary hippocampal cultures were prepared from E18 rat embryos as previously described  and as amyc , mouse monoclonal anti-HA , mouse monoclonal anti-synapsin1 , mouse monoclonal anti-tau1 . The following secondary antibodies were used: goat AMCA-conjugated anti-rabbit , goat Alexa Fluor 488-conjugated anti-mouse IgG2b , goat Alexa Fluor 568-conjugated anti-mouse IgG1 , goat Alexa Fluor 647-conjugated anti-mouse IgG2a .The following primary antibodies were used: rabbit polyclonal anti-c-Fluorescence microscopy was performed using a Zeiss Axioplan2 microscope. All images were acquired with a 63x oil immersion objective (NA 1.4) using MetaMorph imaging software (Molecular Devices). COS-7 cells with similar levels of both surface myc and HA were chosen for imaging and analysis for each co-culture experiment B. Image Constructs of full-length  wild-type human NL3(–A) and mutant NL3(–A) Arg451Cys, fused N-terminally to a Myc and V5 epitope tag, were cloned into the pCAGGS vector, generating pCAGGS-myc-V5-NL3(WT) and pCAGGS-myc-V5-NL3(R451C). Both constructs used native signal sequences and were not C-terminally fused to ECFP. Low density primary hippocampal cultures were prepared from E18 rat embryos as previously described . Neuron myc  and mouse monoclonal anti-V5 . The following secondary antibodies were used: goat Alexa Fluor 488-conjugated anti-rabbit  and goat Alexa Fluor 647-conjugated anti-mouse IgG2a .The following primary antibodies were used: rabbit polyclonal anti-c-Fluorescence microscopy was performed using a Zeiss Axioplan2 microscope. All images were acquired using MetaMorph imaging software (Molecular Devices) and with a 10x air objective to capture the entire neuron in one field of view. The neurons were visualized using the 488 channel for myc and 647 channel for V5.2 and 1% (w/v) Triton X-100 for 2 hr at 277 K. Extracts were centrifuged at 10,000 g for 30 min and the supernatants were diluted 1:1 with extraction buffer. Protein A beads  bound to 100 μg human Fc control protein or MDGA1-, MDGA1ΔIg1-3, or MDGA2-Fc proteins were added and rotated O/N at 277 K. Beads were packed into Poly-Prep chromatography columns (BioRad) and washed with 50 mL of high-salt wash buffer , followed by a wash with 10 mL low-salt wash buffer . Bound proteins were eluted from the beads by incubation with Pierce elution buffer and TCA precipitated overnight. For the MS analysis, we required each protein to have two peptide matches and each peptide to have at least 1 tryptic terminus and an overall protein false discovery rate (FDR) < 1.2% for each dataset. Proteins shown in Affinity chromatography was performed as previously described . BrieflyECTO reported in this paper is PDB: 5OJK. The accession number for the crystal structure of chicken MDGA1ECTO reported in this paper is PDB: 5OJ2. The accession number for the crystal structure of the complex between human NL1(–A–B)ECTO and chicken MDGA1ECTO reported in this paper is PDB: 5OJ6.The accession number for the crystal structure of human NL1(–A+B)All authors contributed to experimental design. J.E., A.J.C., C.H., W.J., and A.C.S. produced constructs and recombinant proteins. A.J.C. and J.E. performed crystallization experiments. J.E., A.J.C., and A.R.A. collected X-ray data and solved the crystal structures. J.E. performed the biophysical experiments. V.C. performed the co-culture and immunocytochemistry experiments. K.M.V., S.N.S., J.N.S., and J.d.W. performed mass spectrometry and pull-down experiments. M.C.R. and H.F. produced the NMDAR samples. J.B., A.M.C., and A.R.A. directed research. J.E. wrote the manuscript, with input from V.C., A.M.C., and A.R.A. All authors provided feedback on the final manuscript version.


In the crystal of the ternary co-crystalline adduct, the components interact through two intermolecular O—H⋯N hydrogen bonds. The supramolecular adducts are interlinked through of halogen bonds and weak non-conventional hydrogen bonds. 3,8]dodecane (TATD)–4-iodophenol (1/2), C8H16N4·2C6H5IO, comprises a half molecule of the aminal cage polyamine plus a 4-iodophenol molecule. A twofold rotation axis generates the other half of the adduct. The components are linked by two intermolecular O—H⋯N hydrogen bonds. The adducts are further linked into a three-dimensional framework structure by a combination of N⋯I halogen bonds and weak non-conventional C—H⋯O and C—H⋯I hydrogen bonds.The asymmetric unit of the title co-crystalline adduct, 1,3,6,8-tetraazatricyclo[4.4.1.1 Non-covalent bonds such as hydrogen bonds (HB) and halogen bonds (XB) attract interest in crystal engineering because they have clear directional properties –4-bromophenol  are < 7%. The asymmetric unit comprises one half of a 1,3,6,8-tetraazatricyclododecane (TATD) molecule and one iodophenol molecule held together by intermolecular O—H⋯N hydrogen bonds  is in good agreement with the value reported for 4-iodophenol itself  and 3.455 (6) Å [C13—H13⋯O1], which exceed the sum of the corresponding van der Waals radii , the strength of the these non-conventional hydrogen bonds can be classified as very weak ° is close to being linear dodecane has already been determined   and 4-iodophenol  was ground at room temperature with a pestle in a mortar for 15 min., as required to complete the reaction (TLC). The mixture was recrystallized from a mixture of n-hexane with a few drops of ethanol to obtain crystals suitable for X-ray analysis, m.p. = 391 K. (yield: 56%).A mixture of 1,3,6,8-tetraazatricyclo[4.4.1.1Uiso(H) set to 1.2Ueq of the parent atomCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017014943/sj5537sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017014943/sj5537Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017014943/sj5537Isup3.cmlSupporting information file. DOI: 1580038CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, QAO anions are stacked in a column along the c axis by π–π stacking interactions [centroid–centroid distances 3.5922 (10)–3.7223 (11) Å]. The columns are interlinked by hexaaquanickel(II) cations through O—H⋯O and N—H⋯O hydrogen bonds.The asymmetric unit of the title compound, [Ni(H Many new quinoline derivatives have been synthesized and used as new potential agents to treat HIV (Cecchetti Q) was synthesized from eugenol and its antibacterial activities have been reported amino]-1-methyl-3-sulfoquinoline (QAO). As polysubstituted quinoline rings are known to coordinate to metal ions, the reaction between QAO and NiCl2 was studied. The reaction product could not be characterized unambiguously by IR or 1H NMR spectroscopy. Although the obtained spectroscopic data are different from those of free QAO, indicating the presence of a deprotonated hydroxyl group, no conclusion about complex formation was possible and further investigation by X-ray diffraction was necessary.The new quinoline derivative (6-hydroxy-3-sulfoquinolin-7-yloxy)acetic acid 6]2+, located about an inversion center  Å] from the best plane through the quinoline ring system (r.m.s. deviation = 0.009 Å). The 2-hydroxyethylamino substituent shows a +sc conformation [torsion angle N18—C19—C20—O21 = 57.0 (2)°].The structure determination shows that Nier Fig. 1. The 6-hQAO molecules running along the c axis through π–π stacking interactions between the quinoline ring systems 2+ complex interacts in total with twelve QAO molecules and two water molecules through O—H⋯O and N—H⋯O hydrogen bonds  was synthesized starting from the natural product eugenol and further transformed to 5-bromo-6-hydroxy-7-[(2-hydroxyethyl)amino]-1-methyl-3-sulfoquinoline (QAO) according to a procedure described by Dinh et al. (2012The quinoline derivative (6-hydroxy-3-sulfoquinolin-7-yloxy)acetic acid  in 10 mL water was added dropwise to 15 mL aqueous solution of QAO  and NH3 (pH ≃ 6–7). The obtained solution was stirred and refluxed at 313–323 K for three h. The brown precipitate was collected by filtration, washed consecutively with ethanol and dried in vacuo. The obtained crystals were soluble in water and DMSO, but insoluble in ethanol, acetone and chloroform. The yield was 60%. Single crystals suitable for X-ray investigation were obtained by slow evaporation from a ethanol–water (1:2 v/v) solution at room temperature.A solution containing NiCl−1): 3510, 3334 ; 3080, 2942 (νC-H); 1588, 1540 (νC=Cring or νC=N); 1190, 1036 , 632 (νC-Br). 1H NMR : 8.34 , 8.27 , 6.51 , 4.22 ; 3.69 ; 3.45 , 7.34 (NH).IR  values were assigned as 1.5Ueq of the parent atoms .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016012408/is5459sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016012408/is5459Isup2.hklStructure factors: contains datablock(s) I. DOI: 1497073CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-017-09784-z, published online 30 August 2017Correction to: This article contains a typographical error in the Conclusions section, where:3 (30 ppm)22 and the lethal 10 minutes HF toxicity value (AEGL-3) is 0.0139 g/m3 (170 ppm)23.”“The immediate dangerous to life or health (IDLH) level for HF is 0.025 g/mshould read:3 (30 ppm)22 and the lethal 10 minutes HF toxicity value (AEGL-3) is 0.139 g/m3 (170 ppm)23.”“The immediate dangerous to life or health (IDLH) level for HF is 0.025 g/m


The conformation about the C=N bond is E. In the crystal, N—H⋯N hydrogen bonds connect pairs of molecules into dimers. In addition, weak C—H⋯O hydrogen bonds and C—H⋯π interactions are observed.The title Schiff base was synthesized  20H27N3O, was synthesized by condensation reaction of 3,5-di-tert-butyl-4-hydroxybenzaldehyde and 2-hydrazinylpyridine, and crystallizes in the centrosymmetric monoclinic space group C2/c. The conformation about the C=N bond is E. The dihedral angle between the rings is 18.1 (3)°. An intermolecular N—H⋯N hydrogen bond generates an R22(8) ring motif. In the crystal, N—H⋯N hydrogen bonds connect pairs of molecules, forming dimers. Density functional theory (DFT) optimized structures at the B3LYP/6–311 G level are compared with the experimentally determined molecular structure in the solid state.The title compound, C The tautomerization in salicylideneanilines has been the subject of particular interest because it is closely related to thermochromism and photochromism. While salicylideneanilines are widely used as precursor compounds for the design of various type new metal complexes, they are also convenient model compounds for studying theoretical aspects of coordination chemistry and photochemistry, as well as for designing molecular architectures by means of molecular motifs capable of hydrogen-bond formation. The present work is a part of an ongoing structural study of Schiff bases and their utilization in the synthesis of quinoxaline derivatives °. The N—N and N—C bond lengths are of 1.396 (7) and 1.253 (7) Å, respectively, indicate single- and double-bond character for these bonds. The C1—O1 bond length of 1.370 (6) Å indicates single-bond character. The conformation about the C15=N1 bond is E with an N2—N1—C15—C4 torsion angle of 177.9 (5)°. Bond distances for (I)et al., 2013tert-butyl groups.The molecular structure of (I)In the crystal, molecules are connected by pairs of N—H⋯N hydrogen bonds Fig. 2, forming0] Fig. 3. There aGAUSSIAN09  level et al., 1998tert-butyl-2,6-diformylphenol hydrazono]methyl}phenol used for stabilization of oxidovanadium(IV)  and 2-hydrazinylpyridine 0.046 g (0.427 mmol) in methanol was refluxed for 3 h in the presence of a catalytic amount of glacial acetic acid. After cooling, the red-coloured precipitate was washed with hot methanol several times, and then dried, giving a red-coloured shiny crystalline compound in 86% yield (0.120 g). Red block-like crystals of the title compound were obtained by slow evaporation of a solution in dichloromethane and ethanol (5:1 v/v).A mixture of 3,5-di-Uiso(H) = 1.2Ueq(C). The N-bound H atom was located in a difference-Fourier map but was also allowed to ride in the refinement with N—H = 0.86 Å and Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017011707/hg5492sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017011707/hg5492Isup2.hklStructure factors: contains datablock(s) I. DOI: 1567740CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


High efficacy and low toxicity are critical for cancer treatment. Polyoxometalates (POMs) have been reported as potential candidates for cancer therapy. On accounts of the slow clearance of POMs, leading to long-term toxicity, the clinical application of POMs in cancer treatment is restricted. To address this problem, a degradable organoimido derivative of hexamolybdate is developed by modifying it with a cleavable organic group, leading to its degradation. Of note, this derivative exhibits favourable pharmacodynamics towards human malignant glioma cell (U251), the ability to penetrate across blood brain barrier and low toxicity towards rat pheochromocytoma cell (PC12). This line of research develops an effective POM-based agent for glioblastoma inhibition and will pave a new way to construct degradable anticancer agents for clinical cancer therapy. Polyoxometalates (POMs) are a large family of metal oxide cluster anions with different applications in magnetics1235678101112141516171819202122232415161820212324262729in vivo and cannot be timely excreted, causing the side effect. Because the slow clearance of POMs can interfere the body’s metabolism and result in the long-term toxicity31via covalent modification3334353616In the medicinal chemistry of POMs, previous works on cancer therapy were mainly focused on the heteropolyacids, including Keggin14192023246O18(≡NC6H4-2-CH3-6-CON(Cy)-CO-NH-Cy)]2− (POM-AMB-acy)   can not 4N]2[Mo6O19], 2-amino-3-methylbenzoic acid and N,N′-dicyclohexylcarbodiimide (DCC) in dry acetonitrile can afford POM-AMB-acy in 23% yield after 20 h, as monitored by ESI-MS (via a Mo ≡ N triple bond with the Mo1-N1 bond length of 1.735 (4) Å and the linear C1-N1-Mo1 bond angle of 176.3 (8)°, which are in great agreement with the typical organoimido groups grafted at an octahedral d0 metal center33A refluxing reaction of 2[Mo6O19], (Bu4N)2[Mo6O18(NC10H15)] (POM-Ad)50 values of ‘6 equals of Na2MoO4·2H2O’ (53.4 μM) was two-folds higher compared to POM-AMB-acy (24.8 μM). This indicated that the functionalized POM, modified with N-acylureido group (acy) and 2-amino-3-methylbenzoxyl group (AMB), has better performance than metal ion itself. Furthermore, the difference between [Bu4N]2[Mo6O19], POM-Ad, and POM-AMB-acy suggested that imidoylization can improve the inhibitory performance of POM towards malignant glioma cells on certain degree.It needs to be figure out whether the synergistic effect originates from POM and organic functional moiety can indeed facilitate the inhibition performance of POM-AMB-acy towards U251 cells. Recently, it is reported that an organic functionalized POM derived from amantadine exhibited better anticancer performance against cancer cells than non-substituted hexamolybdates or amantadine independentlyvia tail vein injection. As shown in −1) for 10 min, the molybdenum level in brain rapidly reached 4.4 mg kg−1, respectively. While in the control group, the molybdenum level was merely 0.2 mg kg−1.To investigate the location of POM-AMB-acy in U251 cells, scanning transmission electron microscopy (STEM) was employed to observe the location of molybdenum, and energy dispersive X-Ray spectroscopy (EDX) was utilized to track the ratio of molybdenum in each area. As shown in in vitro experiments were performed to investigate its degradability by utilizing the fresh cell incubation medium (MEM + 10% FBS) as demonstration. In order to investigate its stability in MEM, IR and ESI-MS was chosen to study its stability. According to −1 compared with 40 min, and it can also be seen in the spectrum of Na2MoO4 in MEM . Recent researches have reported that molybdenum oxide (MoOx) nanosheets could also be degraded into MoO42− in serum, which is corresponding to our result42−.Furthermore, the ESI-MS was also conducted to investigate the composition of degradation product. After maintaining the system  for 24 h, this inorganic degraded product was marked as complex  1460.86 . HoweverT1) on cellular metabolism behavior towards PC12 cell was explored. As shown in 2MoO4·2 H2O’ (94.9%). Therefore, it is deduced that the degradability could alleviate the toxicity of POM-AMB-acy on certain degree. To further investigate the dynamics of degrading process on cytotoxicity of PC12 cells, POM-AMB-acy was pretreated with the cell incubation medium (MEM + 10% FBS) for certain hours, including 0 h, 1 h, 4 h and 22 h, and then added into PC12 cells for further incubation. As shown in T1). Therefore, POM-AMB-acy can be degraded and achieved low toxicity no more than 22 hours. Therefore, via degradation, the toxicity of POM-AMB-acy can be reduced and the long-term toxicity of POM can be alleviated on a certain degree.To understand whether the degradable characteristic is critical to alleviating the toxicity of POM-AMB-acy, POM-AMB-acy itself and degraded POM-AMB-acy complex , and all experiments were carried out in the negative-ion mode.IR spectra were measured by using a Perkin Elmer FT-IR spectrophotometer on KBr pellets in the range of 4000–400 cmKα radiation (λ = 0.71073 Å) at 94(2) K. Data collection, data reduction, cell refinement, and experimental absorption correction were performed with the software package of Rigaku RAPID AUTO . Structures were solved by direct methods and refined against F2 by full matrix least squares. All non-hydrogen atoms, except disordered atoms, were refined anisotropically. Hydrogen atoms were generated geometrically. All calculations were performed using the SHELXS-97 program package.A red single crystal of POM-AMB-acy with three dimensions of 0.40 mm × 0.50 mm × 0.50 mm was selected for diffraction analysis. The data collection was performed on a Rigaku RAXIS-SPIDER IP diffractometer at 50 kV and 20 mA, using graphite monochromatized Mo 2 environment at 37.0 °C. Cells were plated at a density of 1 × 106 cells per 100 mm culture dish and allowed to grow to approximately 70% confluence before experimentation.U251  and PC12 cells (rat pheochromocytoma) were obtained from the Cancer Institute of Chinese Academy of Medical Science  and grown in MEM supplemented with 5% fetal bovine serum and 10% horse serum in a humidified 5% CO5 cells/mL. After 24 h incubation, cells were treated with 1 at the concentration of 60 μM for 24 h. The cells were directly harvested with a cell scraper and centrifuged at 2500 rpm for 10 min. After fixation by a 2.5% (wt/vol) glutaraldehyde and 2% (wt/vol) paraformaldehyde, samples were then submitted to the Center of Biomedical Analysis (Tsinghua University) for subsequent treatment and sent to Analysis and Test Center (Tsinghua University) for STEM and EDX analysis. Of note, in order to clearly identify the distribution of molybdenum in cancer cells using STEM mode, it had to give up using osmium tetroxide for fixation, so the morphology of U251 cells lost on a certain degree.U251 cells were seeded in 10 cm dishes at a density of 1 × 104N)4[Mo8O26] , amantadine hydrochloride  and DCC  were added into 10 mL anhydrous acetonitrile and refluxed under dry N2 for 9 hours. During the reaction procedure, reactants gradually dissolved and the color of the solution turned into light green. By cooling it down to room temperature, the white precipitate  was moved by filtration. With the slow evaporation of acetonitrile from the filtrate, the yellow block crystals appeared . Elemental analysis Calc (%) for C46H93Mo6N5O18 (M = 1579.89): C, 34.94; N, 4.43; H, 5.89. Found: C, 34.88; N, 4.39; H, 5.85. IR : 2961, 2932, 2874, 1481, 1380, 1236, 973, 944, 783 (absorbance at 973 is characteristic peak for mono-organoimido substituted hexamolybdate). UV/vis : λmax = 325. ESI-mass spectrometry : 1254.7 , 1015.4 , and 508.2  were assigned to [Bu4N][Mo6O18N(C10H15)]−, [HMo6O18N(C10H15)]− and [Mo6O18N(C10H15)]2−, respectively.According to the previous literature4N)2[Mo6O19] , 2-amino-3-methylbenzoic acid  and DCC  were added into 15 mL anhydrous acetonitrile at 110 °C under the protection of dry N2. During the reaction, the color of the solution gradually changed from orange to red then the dark red. After 20 hours, by cooling to room temperature, solution was filtrated to remove the white precipitate . Slowly evaporating the filtrate in open air, some sticky oil was obtained. Then it was carefully washed by toluene and ether for several times. By the slow gas-phase diffusion of ether into the filtrate, POM-AMB-acy was deposited as red crystals within several days. Yield of (Bu4N)2[Mo6O18(≡NC6H4-2-CH3-6-CON(Cy)-CO-NH-Cy)]: 0.78 g, 23%, based on Mo. Elemental analysis (%) calcd for Mo6O20C53N5H102 (M = 1705.05 g mol−1): C 37.33, N 4.11, H 5.98; found: C 37.40, N 4.08, H 5.98; IR : 2961 (m), 2933 (m), 2874 (w), 1482 (w), 1380 (w), 976 (sh), 951 (sh), 783 (sh) (the band at 976 is a characteristic peak for mono-organoimido-substituted hexamolybdate); UV/vis : λmax = 351; ESI-MS : 1460.86 , 1219.58 , and 609.28 , assigned to [Bu4N][Mo6O18(≡NC6H4-2-CH3-6-CON(Cy)-CO-NH-Cy)]−, [HMo6O18(≡NC6H4-2-CH3-6-CON(Cy)-CO-NH-Cy)]−, and [Mo6O18(≡NC6H4-2-CH3-6-CON(Cy)-CO-NH-Cy)]2−, respectively.A mixture of (Bu4H10O): Mo6O21C57N5H110, M = 1777.14, Monoclinic, space group P21/c, a = 11.7348(14) Å, b = 25.846(2) Å, c = 23.951(3) Å, α = 90°, β = 93.220(13)°, γ = 90°, V = 7252.8(14) Å3, T = 94 K, Z = 4, Dc = 1.628 g cm−3, m = 1.076 mm−1, GooF = 1.088, Final R indices (I ≥ 2σ(I)) R1 = 0.0755, wR2 = 0.1559.Crystal data for compound (POM-AMB-acy) for corresponding period  at the concentration of 80 μM. After 24 h of incubation, the cells were treated with 100 μL of cell medium and 100 μL of mixture solution which contains the pretreated POM-AMB-acy, so the final concentration of POM-AMB-acy was 40 μM and the cells were incubated for another 24 h. For each test, five replicates were employed. Twenty microliters of 5 mg/mL MTT solution were added to each well, and cells continued to be incubated for 4 h at 37.0 °C. After careful removal of the medium, dimethyl sulfoxide (DMSO) was added to each well, and the plate was then shaken for about 10 min. Absorbance was then measured at 490 nm in a microplate reader . The curves of viability were drawn by comparing the control group. The inhibitory rate was calculated using the following equation: inhibitory rate (%) = (ODcontrol − ODtreatment)/ODcontrol × 100%. The viability rate (%) = 100 − inhibitory rate (%).To evaluate the cytotoxicity of degraded POM-AMB-acy complex, the PC12 cells were seeded at density of 5 × 10How to cite this article: She, S. et al. Degradable Organically-Derivatized Polyoxometalate with Enhanced Activity against Glioblastoma Cell Line. Sci. Rep.6, 33529; doi: 10.1038/srep33529 (2016).


S(5) and S(6) rings. In the crystal, weak C—H⋯O hydrogen bonds link the molecules into a three-dimensional network.In the title compound, the quinoline ring system forms a dihedral angle of 20.9 (1)° with ethynyl-substituted benzene ring. The unsubstituted phenyl ring forms a dihedral angles of 52.7 (1)° with the quinoline ring system and 54.1 (1)° with the ethynyl-substituted benzene ring. An intramolecular bifurcated N—H⋯ hydrogen bond forms  25H16N2O2, the quinoline ring system is essentially planar, with a maximum deviation of 0.030 (1) Å, and forms a dihedral angle of 20.9 (1)° with benzoyl benzene ring. The unsubstituted phenyl ring forms dihedral angles of 52.7 (1)° with the quinoline ring system and 54.1 (1)° with the ethynyl-substituted benzene ring. The molecule contains an intramolecular bifurcated N—H⋯ hydrogen bond, forming S(5) and S(6) rings, which may influence the conformation of the molecule. In the crystal, weak C—H⋯O hydrogen bonds link the molecules into a three-dimensional network. In addition, the three-dimensional structure contains π–π stacking interactions, with centroid–centroid distances of 3.695 (1) and 3.751 (1) Å.In the title compound, C The biological activity of these ligands can be attributed to distinct chemical and biochemical advantages: they are chemically more stable than diazo esters, aryl azides and diazirines, and can be manipulated in ambient light and can be activated at 350–360 nm, avoiding protein-damaging wavelengths. These properties produce highly efficient covalent modifications of macromolecules, frequently with remarkable specificity  and S(6) rings, which may influence the conformation of the molecule.The molecular structure of the title compound is shown in Fig. 1Cg1⋯Cg2 and 3.751 (1) Å for Cg3⋯Cg3 where Cg1, Cg2 and Cg3 are the centroids of the C11–C16, C20–C25 and C1–C6 rings, respectively.In the crystal, weak C—H⋯O hydrogen bonds Table 1 link theet al., 2016et al. 2008et al. 2012N-(2-benzoylphenyl)quinoline-2-carboxamide moieties are reported  and benzonitrile (3 equiv) for 24 h at approximately 353 K. The solution was extracted with DCM, dried and concentrated to obtain (2-amino-4-bromophenyl)(phenyl)methanone (2) . Compound 2 (1.8 mmol) was dissolved in triethylamine, Pd(PPh3)2Cl2 (0.05 eq), trimethylsilylacetylene (1.5 eq) and copper iodine (0.1 eq) were added and the solution was heated to approximately 343 K overnight. The organic phase was separated and concentrated  and the fraction containing the product (75%) was collected and used for the next step. A solution of compound 3  in tetrahydrofurane was stirred and cooled in an ice bath, tetra-n-butylammonium fluoride (1.5 eq) was added and the reaction was stirred for two hours. The organic layer was separated and dried over magnesium sulfate to obtain compound 4 . The title compound (I)p-toluenesulfonyl chloride and compound 4 for 24 h in dichloromethane. After evaporation of the CH2Cl2, the compound was purified by silica column chromatography . Single colourless block-shaped crystals of (I)The title compound was prepared using 3-bromoaniline 1, Fig. 3 as startN-(2-benzoyl-5-ethynylphenyl)quinoline-2-carboxamide (I) Colourless solid . 1H NMR : δ 9.11 , 8.41 , 7.89 , 7.84 , 7.67 , 7.60 , 7.50 , 7.28 , 3.27 . 13C NMR : δ 198.0 (Cquat), 163.7 (Cquat), 149.6 (Cquat), 146.6 (Cquat), 139.7 (Cquat), 138.6 (Cquat), 137.6 (Cquat), 133.1 (+), 132.5 (+), 130.5 (+), 130.2 (+), 129.9 (+), 129.4 (+), 128.3 (+), 127.6 (+), 125.8 (+), 124.8 (+), 118.4 (+), 82.8 (Cquat), 80.3 (+).Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017004601/lh5838sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017004601/lh5838Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017004601/lh5838Isup3.cmlSupporting information file. DOI: 1539719CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II ion has a distorted octahedral RuN2O4 coordination environment defined by two 3,5-di-tert-butyl-o-benzosemiquinone ligands and one 2,2′-bipyridine ligand. In the crystal, the compounds are linked by C—H⋯O and π–π stacking interactions, resulting in a layer structure.In the title compound, the Ru 14H20O2)2(C10H8N2)], the RuII ion has a distorted octahedral coordination environment defined by two N atoms of the chelating 2,2′-bipyridine ligand and four O atoms from two 3,5-di-tert-butyl-o-benzoquinone ligands. In the crystal, the complex molecules are linked by intermolecular C—H⋯O hydrogen bonds and π–π stacking interactions between the 2,2′-bipyridine ligands [centroid–centroid distance = 3.538 (3) Å], resulting in a layer structure extending parallel to the ab plane.In the title mononuclear complex, [Ru(C It has been reported that o-quinone derivatives are non-innocent electroactive ligands that can be found as neutral quinones, radical semiquinones or dianionic catecholates  ligand, namely [Ru(bpy)(C6H4O2)2] and [Ru(bpy)(C14H20O2)2] (title compound), have been investigated by using various experimental techniques  species, it was necessary to oxidize the complexes by silver perchlorate in non-aqueous media. Lever et al. (1988II(bpy)(sq)2  with significant mixing of metal and ligand orbitals through Ru–sq π back-donation, which results in elongation of the C—O bonds of o-quinone ligands. This elongation has been demonstrated for [Ru(bpy)(C6H4O2)2] by X-ray single crystal analysis, but the structure of the title compound has not previously been characterized.The coordination chemistry of  al. 1988 concludeII ion has a distorted octahedral RuN2O4 coordination environment defined by two 3,5-di-tert-butyl-o-benzosemiquinone anion-radicals and one 2,2′-bipyridine ligand 2] .A search of the Cambridge Structural Database  was added to 20 ml dry methanol and then triethylamine  was added dropwise and the resultant mixture was stirred for 5 min. Ru(bpy)2Cl2  was then added to the solution and the contents were refluxed for 6 h. After refluxing, the reaction mixture was cooled down to room temperature and the contents were filtered off. The obtained residue was washed with cold methanol and dried in vacuo . Slow evaporation of a solution of the compound in a CH2Cl2–MeOH mixture  yielded single crystals suitable for X-ray diffraction. Crystals of title compound gave no EPR signal at room and liquid nitrogen temperatures, and thus are diamagnetic.3,5-Di-Uiso(H) = 1.5Ueq(C). The remaining (aromatic) H atoms were placed geometrically and refined using a riding model, with C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901700281X/is5467sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901700281X/is5467Isup2.hklStructure factors: contains datablock(s) I. DOI: 1533648CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, molecules are linked  21H14N2O2, was prepared by Pictet–Spengler cyclization of tryptamine and 4-formyl coumarin. In the molecule, the dihedral angle between the mean planes of the coumarin and β-carboline ring systems is 63.8 (2)°. In the crystal, molecules are linked via N—H⋯N hydrogen bonds, forming chains along the b-axis direction. Within the chains, there are a number of offset π–π interactions present [shortest intercentroid distance = 3.457 (2) Å].The title compound, C However, only six of these structures contain the 7-methyl-4-phenyl-2H-chromen-2-one nucleus . There were no structures reported for a search of 7-methyl-4-(pyridin-2-yl)-2H-chromen-2-one skeleton.A search of the Cambridge Structural Database  to give the intermediate. This intermediate compound (1 eq) was taken in 10 ml of dry chloroform and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2 eq) was added at intervals of 5 min in cold conditions, 273 K. Stirring was continued for ca 10 h. The reaction mixture was then quenched using aqueous sodium bicarbonate and extracted with chloroform. The organic layer was washed 2–3 times with sodium bicarbonate, water and brine solution, dried using sodium sulfate, and concentrated to afford the crude title product. It was purified by flash chromatography using 230–400 mesh silica-gels . The solid obtained was recrystallized from dichloromethane, giving colourless block-like crystals of the title compound on slow evaporation of the solventAcetic acid (10 ml) was added drop wise, at 273 K, to a mixture of tryptamine (1 eq) and 4-formyl coumarin (1 eq). The reaction mixture was stirred at room temperature for Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016019769/su5339sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016019769/su5339Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016019769/su5339Isup3.cmlSupporting information file. DOI: 1522101CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


P-helimer structure incorporating π–π and/or σ–π interactions.In the title compound, the zinc(II) atom exhibits a a distorted five-coordinate square-pyramidal geometry and is coordinated by one trifluoromethanesulfonate ligand and four N-donor atoms. The resulting complex shows a single-stranded  48H31N4)(CF3SO3)](CF3SO3)·1.5CH2Cl2, adopts a distorted five-coordinate square-pyramidal geometry. It is coordinated by one trifluoromethanesulfonate ligand and four N atoms of the N2-[(benzo[h]quinolin-2-yl)methyl]-N2′-[(benzo[h]quinolin-2-yl)methylidene]-1,1′-binaphthyl-2,2′-diamine ligand. The complex is present as a single-stranded P-helimer monohelical structure incorporating π–π and/or σ–π interactions. One of the imine bonds present in the original ligand framework is reduced, leading to variations in bond lengths and torsion angles for each side of the ligand motif. The imine-bond reduction also affects the bond lengths involving the metal atom with the N-donor atoms located on the imine bond. There are two molecules of the complex in the asymmetric unit. One of the molecules exhibits positional disorder within the coordinating trifluoromethanesulfonate ion making the molecules symmetrically non-equivalent.The zinc(II) atom in the title compound, [Zn(C Stereochemistry plays a very important role in the chemical interactions that dominate several fields of chemistry . The ZnII cation is coordinated by four N-donor atoms from the N2-[(benzo[h]quinolin-2-yl)methyl]-N2′-[(benzo[h]quinolin-2-yl)methylidene]-1,1′-binaphthyl-2,2′-diamine (BQMB) ligand and one triflate anion in a distorted square-pyramidal geometry  and 0.003 (4), respectively, indicating that the absolute structure was unequivocally established. Anomalous dispersion was used to determine the absolute structure. There are two molecules of complex (1) in the asymmetric unit. As seen in Fig. 31). The two molecules of (1) in the asymmetric unit propagate along the b-axis direction via the twofold screw axis. The counter-ions and the solvent molecules fill the void spaces between symmetry-related asymmetric units.The molecules of the crystal structure are related only by a twofold screw axis running along the on Fig. 2. The reset al., 20165 values of 0.86 and 0.93), two structures have a square-pyramidal geometry , and the last structure assumes a distorted square-pyramidal geometry as evidenced by the τ5 value of 0.48. The Zn—O bond length for (1) falls on the shorter end of the distance spectrum. Meanwhile the Zn—N distances for three of the contacts agree well with those in the previously reported structures. The fourth contact at a distance of 2.253 (4) Å falls above the average Zn—N distance by 0.176 Å, presumably due to the greater flexibility within the ligand framework resulting from the imine reduction.The survey of Cambridge Structural Database (Groom 1) is given in Fig. 4The synthetic scheme for (Synthesis of (R)-N,N′-bis[(2-benzo[h]quinolinyl)methylene]-2,2′-diamine (BQMB) ligand: the BQMB ligand was synthesized following established literature procedures --2,2′-diamine  and 2-formylbenzoquinoline  were refluxed in ethanol (25 ml) for 2 h. A yellow precipitate was obtained, which was filtered and washed twice with 10 ml aliquots of ethanol. The resulting yellow mixture was dried under vacuum for 30 minutes to afford BQMB as a yellow solid . 1H NMR : 7.32 , 7.35 , 7.40 , 7.46 , 7.55 , 7.58 , 7.63 , 7.72 , 7.77 , 7.81 , 8.01 , 8.07 , 8.10 , 8.70 , 8.80 . 13C NMR : δ 119.16, 119.33, 124.39, 125.54, 125.76, 127.05, 127.35, 127.46, 127.62, 128.19, 128.34, 128.56, 128.67, 129.23, 130.04, 131.68, 132.76, 134.01, 134.04, 136.67, 146.35, 148.72, 153.89, 162.82. Elemental analysis for (C48H30N4): calculated C 86.98, H 4.56, N 8.45; found C 86.97, H 4.85, N 8.45.Synthesis of {(R)-N2-[(benzo[h]quinolin-2-yl)methyl]-N2′-[(benzo[h4]quinolin-2-yl)methylidene]-1,1′-binaphthyl-2,2′-diamine-κN,N′,N′′,N′′′}(trifluoromethanesulfonato-κO)zinc(II)} trifluoromethanesulfonate dichloromethane 1.5-solvate: the BQMB ligand  was dissolved in a mixture of 15 ml ethanol and 10 ml tetrahydrofuran in a 100 ml round-bottom flask. Sodium borohydride  and zinc(II) trifluoromethanesulfonate  were added to the flask to give an orange-colored solution. The reaction was allowed to reflux for 15 h and then cooled, producing a reddish-orange-colored precipitate which was filtered and washed twice with a cold solvent mixture. The precipitate was dried under vacuum for 30 minutes to yield a reddish-orange-colored solid . Reddish-orange-colored single crystals suitable for X-ray analysis were obtained by slow solvent diffusion of hexane into a concentrated complex solution in dichloromethane.A were refined at idealized positions and allowed to ride on neighboring atoms with relative isotropic displacement parameters. The amine hydrogen atoms were refined as riding freely. The asymmetric unit contains two molecules of (1), two triflate counter-ions, and three molecules of dichloromethane solvent. One of the molecules of (1) exhibited positional disorder within the coordinating triflate ion. The positional disorder was modeled over two positions with the major component contributing 88.1 (4)%. Due to the low occupancy of the minor component, idealized geometry was used to stabilize the refinement and the component was refined isotropically. Additional disorder was observed in one of the solvent molecules of dichloromethane. Two positions were used to model the positional disorder and the major component refined to occupancy of 50 (4)%. The bond lengths C54A—Cl5A and C54A—Cl6A were restrained to be similar.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989017008027/im2476sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017008027/im2476Isup2.hklStructure factors: contains datablock(s) I. DOI: 1553147CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Bacillus spp. was searched for in methanol (MeOH) extracts of soybean curd residues, and diacetonamine (1) was identified as the active compound. SIF was first isolated as a monoacetylated derivative , and its chemical structure was elucidated by field desorption mass spectrometry, electron ionization mass spectrometry, and nuclear magnetic resonance (NMR) analyses. After 48-h incubation, 40 µM diacetonamine hydrochloride (1b) exhibited sporulation-inducing activity with 35% sporulation frequency toward a Bacillus amyloliquefaciens wild-type strain (AHU 2170), whereas 40 µM diacetone acrylamide (3) showed 99% sporulation induction, which was much higher than that of 1b. Although Bacillus megaterium NBRC 15308 was sporulated by the treatment with 400 µM 1b with 36 and 70% sporulation frequency after 72- and 96-h incubation respectively, 3 at the same concentration showed only 2% sporulation after 72-h incubation. Hence, diacetonamine (1) was characterized as a genuine SIF from soybean curd residues, but it was uncertain whether 1 is a natural product or an artifact. Spores of B. amyloliquefaciens induced by 1b survived after treatment with heating at 95 °C for 10 min, also suggesting that 1 is genuine SIF in soybean curd residue. As sporulation induction is likely linked to activation of antibiotic production in some spore-forming Firmicutes bacteria, compound 1 would be a possible chemical tool to develop an effective fermentation technology in Bacillus species.Under bioassay-guided investigation, a sporulation-inducing factor (SIF) toward The online version of this article (doi:10.1186/s13568-017-0395-0) contains supplementary material, which is available to authorized users. Firmicutes, such as Bacillus, Paenibacillus, and Clostridium, form endospores under nutrient-starvation conditions, an event known as sporulation  at 270/67.5 MHz and a Bruker AMX-500  at 500/125 MHz.Electron ionization mass spectrometry (EI-MS), field desorption mass spectrometry (FD-MS), and field desorption high-resolution mass spectrometry (FD-HR-MS) were conducted using MS spectrometers JMS-T100GCV and JMS-SX-102 . 1a) was purchased from Sigma-Aldrich , and diacetonamine hydrochloride (1b) and N-acetyldiacetonamine (2) were prepared from 1a. As another chemical derivatizable from 1, diacetone acrylamide (3) was purchased from Wako , while a β-amino acid, 3-amino-3-metylbutyric acid (4), used as a negative control was purchased from Combi-Blocks . Their chemical structures are shown in Fig. Diacetonamine hydrogen oxalate (tonamine  were preB. amyloliquefaciens AHU 2170 isolated from the rhizosphere of Chinese cabbage (Brassica rapa var. pekinensis) and stocked in our laboratory  agar plate before use. The B. amyloliquefaciens wild-type strain easily formed spore-based pellicle in NB liquid medium, whereas B. megaterium was a rarely sporulating Bacillus species  at 30 °C for 48 h in the dark as reciprocation of 100 rpm, the samples were divided into four portions  and centrifuged at 3500g at 4 °C for 15 min. The resulting bacterial cell pellet was re-suspended in 10 mL MSG  medium , and a 10 µL portion of the solution was added to 10 mL MSG medium previously autoclaved. The 10-mL cell suspension was linear-shaken in a 30-mL Erlenmeyer flask at 80 rpm at 30 °C for 48 h for B. amyloliquefaciens. B. megaterium required 72–120 h for sporulation.After shaking culture of g for 1 min to remove the supernatant. The resulting cell pellet was re-suspended in 100 or 20 µL Milli-Q water, and a 3-µL portion of the resulting cell suspension was dropped on a glass slide  and pressed tightly with a cover glass (Matsunami Glass). The prepared slide was observed using a fluorescent microscope BioRevo  under 1500× magnification with an oil immersion lens.A LIVE/DEAD BacLight Bacterial Viability Kit (L13152)  distinguishable from vegetative cells . Using a GFP-BP filter , live cells and spores were stained as green with SYTO 9, whereas propidium iodide with a TRITC filter  stained dead cells red. Competent cells , while sporulated cells were more spherical endospores (approximately 0.8 µm × 1.0 µm) than those of B. amlyloliquefaciens and easily distinguishable from vegetative cells. Immature spores of B. megaterium were stained by SYTO 9, while mature spores were not.Vegetative cells of 1–105-fold with Milli-Q water in a polymerase chain reaction (PCR) tube was heated at 95 °C for 10 min in a PCR thermocycler. After cooling to room temperature, the suspension was well mixed by pipetting and spread over a NB agar plate using a disposable bacteria spreader. After 24-h incubation at 30 °C in the dark, colonies were counted. As a blank, all bacterial cell suspensions were spread on NB agar plates without heating treatment.A 100-µL portion of spore suspension diluted 10n-BuOH) . The resulting aqueous layer was the only fraction active as SIF.A 10-kg fresh weight soybean curd residue  was collected from grocery shops and mixed for extraction with methanol (MeOH). The soybean curd residues were soaked in 10 L of MeOH, and the methanolic extract was collected by decantation and squeezing the remaining residues in a cotton cloth. The squeezed residues were re-extracted with another 6 L of MeOH. The methanolic extracts further collected and combined were filtered in vacuo and concentrated under a low pressure to 500 mL of an aqueous solution. This aqueous solution was partitioned with hexane , ethyl acetate (EtOAc) , and normal butanol  was subjected to gel filtration using a 1.5 L Sephadex G-25  column . Sporulation-inducing activities were observed in the first three fractions, from Fr-G-1 to Fr-G-3. The remaining half portions of the extracts were also subjected to gel filtration at the same scale as the Sephadex G-25 column, and all the active fractions obtained were combined .B. amyloliquefaciens.Fraction Fr-G-1–3 was further fractionated by column chromatography using a 450-mL Sephadex LH-20  column by eluting with 5% MeOH , and Fr-LH-2 and LH-3  showed active sporulation induction toward For cation-exchange chromatography, Fr-LH-2–3 containing active components were applied to a 600 mL-bed volume of a CM-cellulose  open column, previously conditioned with 0.2 M aq. HCl followed by washing with Milli-Q water. The active component was eluted with Milli-Q water in the second fraction  obtained as 4.08 g of syrup.2CO3 solution. As the concentrated solutes  exhibited sporulation induction, all solutes were subjected to a second gel filtration step in a 400-mL Sephadex G-25 column for elution with Milli-Q water (100 mL for each fraction). The third fraction  showed sporulation-inducing activity.Anion-exchange column chromatography using DEAE-cellulose  was subsequently conducted for Fr-CMC-2. The DEAE-cellulose column, previously conditioned with 0.2 M aq. NaOH solution followed by washing with Milli-Q water, retained the active component in the column, despite elution with Milli-Q water (1200 mL). Therefore, the column was continuously eluted with 600 mL of 0.2 M Nan-BuOH:water (H2O):acetic acid (AcOH) 4:1:1. The main substance in the fraction gave a dark brown-colored spot at Rf 0.4 by spraying with vanillin-sulfuric acid reagent. The main compound was obtained by preparative TLC (10 × 10 cm) in n-BuOH:H2O:AcOH 4:1:1 for a small portion of Fr-DS-3. However, the main compound obtained and eluted from silica gel with 50% MeOH showed no sporulation induction. The chemical substance recovered from the plate lower zones of the main band also eluted with 50% MeOH and showed clear sporulation activity.Fraction Fr-DS-3 was analyzed by normal phase silica gel thin-layer chromatography (TLC)  developed in p-anisaldehyde-sulfuric acid reagent followed by heating, but this compound showed a severe tailing in the acidic solvent. Thus, we replaced AcOH with 25% aq. NH4OH solution, and the substance gave a fine spot on normal phase silica gel TLC in n-BuOH:25% aq. NH4OH:acetone 4:5:2.This fraction exhibited a characteristic unique pinkish color by spraying with n-BuOH: 25% aq. NH4OH: acetone (4:5:2). Silica gel column was first conditioned with Milli-Q water followed by the eluting solution, and then Fr-DS-3 was loaded to obtain fractions (25 mL each). Each fraction was immediately concentrated in vacuo. The target substance responsive to p-anisaldehyde-sulfuric acid reagent was detected in Fr-15–Fr-22 as a single spot on TLC. All fractions (SIF-Fr-15-22) together (450 mg of a colorless solid) exhibited significant sporulation-inducing activity at a concentration equivalent to 1 g soybean curd residue per mL medium (45 µg SIF-Fr-15-22 per mL).The active component in Fr-DS-3 (1.00 g) was further purified by silica gel column chromatography  and eluted with m/z, %) of SIF-Fr-15–22 showed several parent ion peaks, such as 407.0 (42), 385.0 (20), 308.1 (21), 215.0 (25), 193.0 (100), and 116.1 (81). We also tested several coloring reagents for the detection of active spots on TLC, including p-anisaldehyde-sulfuric acid reagent and ninhydrin. The target compound showed a pale pink color on spraying with p-anisaldehyde-sulfuric acid reagent followed by strong heating, but no response to ninhydrin.FD-MS  as a purple-colored spot by spraying with p-anisaldehyde-sulfuric acid reagent. This product was purified by preparative TLC developed twice in hexane:EtOAc 1:1 to yield 4.0 mg of colorless syrup. EI-MS : 157 , 142 , 114 , 100 , 98 (42), 83 (57), 72 (24), 60 (22), 58 (100), and 43 (85). 1H-NMR : 5.8 , 2.94 , 2.12 , 1.90 , and 1.39 . 13C-NMR and distorsionless enhancement by polarization transfer (DEPT) : δC 208.18 (CO), 170.11 (COCH3), 52.15 (C), 50.93 (CH2), 31.77 (CH3), 27.52 (CH3 × 2), and 24.36 (COCH3).Although the active component was highly concentrated in SIF-Fr-15–22, the active fraction contained a relatively high amount of silica gel and contaminants. To remove inactive contaminants, a portion of the dried active fraction was subjected to an acetylation reaction. A portion of SIF-Fr-15–22 (29.5 mg) re-dissolved in 2 mL of acetic anhydride and pyridine (1:1) was incubated at 70 °C for 1 h. One of the main products, 1a, Sigma-Aldrich) was subjected to an anion exchange reaction. Compound 1a  dissolved in 10 mL Milli-Q water was passed through a conditioned DEAE cellulose column (200 mL bed volume). An alkaline fraction eluted first was collected and neutralized using 1 M aq. HCl solution and then concentrated to yield 0.82 g of diacetonamine hydrochloride (1b) as a colorless syrup (90% yield).Commercially available diacetonamine hydrogen oxalate (1a (196.4 mg) dissolved in a 1:1 mixture of Ac2O and pyridine (4 mL) was incubated at 70 °C for 1 h. A major product, N-acetyldiacetonamine (2), was obtained by preparative TLC  as a pinkish purple-colored spot upon spraying with p-anisaldehyde-sulfuric acid spray reagent followed by heating.For the acetylation reaction, 1b) was preliminarily tested at concentrations of 4, 40, and 400 µM for sporulation-inducing activities toward B. amyloliquefaciens and B. megaterium. All the test compounds, including 1b, N-acetyldiacetonamine (2), and diacetone acrylamide (3), were dissolved in DMSO to be 400 mM, and diluted with MSG medium into final concentrations of 4, 40, and 400 µM. The MSG medium for the sporulation assay always contained 0.1% DMSO.Diacetonamine hydrochloride  gave one major spot on normal phase silica gel TLC (n-BuOH:H2O:AcOH 4:1:1). However, this acidic and oligomeric sugar-like spot was inactive.We attempted cation- and anion-exchange column chromatography for Fr-LH-2–3. By elution with Milli-Q water, the active substance was completely passed through the cation exchange CM-cellulose column, but elution of the active component was clearly retarded from the void fraction . The active fraction Fr-CMC-2 was further applied to the anion exchange DEAE-cellulose column. By elution with Milli-Q water, the active substance was captured in the column and eventually eluted with 0.2 M Nap-anisaldehyde-sulfuric acid, was active in the sporulation assay. We further focused on this substance obtained as a fine condensed purple spot on TLC in n-BuOH:25% aq. NH4OH:acetone 4:5:2. All Fr-DS-3 was subjected to silica gel column chromatography by eluting with the same solvent as used for TLC. The fractions (Fr-15–22) containing the target substance were combined . This fraction yielding the target compound as a single spot on TLC (n-BuOH:25% aq. NH4OH:acetone 4:5:2) showed clear sporulation induction activity. Approximate calculation by the bioassay suggested that at least 1/10 of the active substance was recovered from the extracts of 10 kg soybean curd residue.In contrast, a weak purple lower spot on TLC, appearing on spraying with Rf 0.15 in hexane:EtOAc 1:1 showed a pinkish color by spraying with p-anisaldehyde-sulfuric acid reagent. This target product  showed its parent ion at m/z 157 (115 + COCH2), indicating a monoacetylated product derived from compound with a molar mass of 116.1 .FD-MS analysis of SIF-Fr-15–22 showed several parent ion peaks, indicating that this active fraction was a mixture. A portion of the active component (29.5 mg) was subjected to acetylation. After this reaction, SFI-Fr-15–22 yielded two major products, and the one detected at 1H-, 13C-NMR, and DEPT spectra of the monoacetylated derivative (1c) in chloroform-d showed two singlet methyl proton signals at δH 1.39 magnetically equivalent , singlet methyl proton signal at δH 1.90 assignable as the acetyl group (–NH–CO–CH3), another singlet methyl proton signal at δH 2.12 assignable as connected with the carbonyl carbon on the skeleton (–CO–CH3), and set of methylene proton signals equivalent at δH 2.94 .13C-NMR and DEPT showed two equivalent methyl carbons at δC 27.52 (×2), methyl carbon next to the carbonyl group (δC 31.76), and methylene carbon at δC 50.93. A quaternary carbon at δC 52.15 was assigned as connected with an amino group and two methyl groups. Carbon signals at δC 170.11 (carboxy carbon) and δC 24.36 (methyl carbon) were assignable to an acetyl group. Thus, one remaining carbon for the chemical formula (C6H13NO + COCH2) was assigned as a carbonyl carbon (δC 208.18) positioned between the methyl carbon (δC 31.77) and methylene carbon (δC 50.93).N-acetyl-4-amino-4-methyl-2-pentanone . Therefore, the compound was identified as diacetonamine  with a molar mass of 115.1 g/mol. From commercially available diacetonamine hydrogen oxalate (1a), N-acetyldiacetonamine (2) was also prepared (yield 89% from 1a) and completely matched with 2 prepared from SFI-Fr-15–22 in the 1H- and 13C-NMR spectra.The monoacetylated compound, derivatized from the active principle, was thus elucidated as 1b), the sporulation-inducing activity of 1 was examined. As our isolation process for compound 1 allowed approximate estimation that 1 kg soybean curd residue contains at most 45 mg (approximately 400 µmol) of 1. Hence, 400 µM was initially set at the concentration of pure 1b tested for sporulation induction toward the vegetative cells of B. amyloliquefaciens. Within 48 h of incubation, compound 1b at 400 µM showed sporulation inducing activity (51% frequency) nearly equivalent to that of the final active fraction from soybean curd residue, but its 1/10 concentration (40 µM) was less active (35% sporulation frequency)  and diacetone acrylamide (3) more stable than 1 were also tested for sporulation induction assay. Toward B. amyloliquefaciens incubated for 48 h, 400 µM of 2 and 3 showed 54 and 69% sporulation frequencies respectively, which were almost equivalent to or obviously higher than that of 1b. Interestingly, 40 µM of 3 showed 99% sporulation without cell death  was observed at 72-h incubation, in which spores were uniquely stained with SYTO 9 as green fluorescence , while 400 µM of 1b induced 70% sporulation to B. megaterium under the same incubation conditions. Conversely, 1b at 4 µM did not induce any sporulation at 96 h or even after the incubation for 120 h  was also examined its sporulation inducing activity toward B. megaterium. Compound 3 at 400 µM did not induce any sporulation at 72-h incubation   was the main active component in soybean curd residue. Isolation and identification of 1 as SIF were successfully achieved due to our simple and quick bioassay system using the B. amyloliquefaciens wild-type strain. In this study, diacetonamine hydrochloride (1b) showed greater than 34 and 51% sporulation frequencies toward vegetative cells of B. amyloliquefaciens at 40 and 400 µM respectively, while 40 µM of diacetone acrylamide (3) induced nearly 100% sporulation frequency in the same sporulation assay. Conversely, vegetative cells of B. megaterium upon exposure to 400 µM of 1b needed more than 72-h incubation for sporulation induction. As B. megaterium is a Bacillus species difficult to induce sporulation  that has been produced on an industrial scale polymerizes with a wide variety of vinyl compounds for a crosslinking agent was characterized as a stable sporulation inducer. Since 3 is water-soluble and more stable than 1, amide derivatives including 3 would be a promising target for a photoaffinity probe design to detect sporulation signal-sensing proteins linked to the sporulation (Asayama and Kobayashi 1) as a SIF-like compound may contribute to rapid preparation of probiotic materials and exploiting an effective fermentation technology for antibiotic production by Bacillus species (Lecca et al. Firmicutes bacteria will be reported elsewhere.As a report on biological activity of


In the crystal, the planar complex molecules are stacked parallel to the c axis, resulting in a columnar structure. In a column, an infinite almost straight Pt⋯Pt chain is formed, suggesting weak metal–metal interactions [Pt⋯Pt = 3.3475 (8) Å]. The crystal packing is stabilized by a three-dimensional N—H⋯Cl hydrogen-bonding network between the amino groups and the Cl ligands of adjacent molecules.The Pt Since the discovery of the antitumor activity of cis-platin (NH2)2}] reported previously (NH2)2}] molecule, the other half being completed by application of twofold rotation symmetry. The PtII atom is coordinated by two N atoms of an 1,2-phenylenediamine ligand and by two Cl− ions in a slightly distorted square-planar configuration cis-[PtCl2(NH3)2] ] 2] ]·0.33-hydrate (NH3)] ] ] ] ] 2]Cl2·2H2O  units are arranged in parallel and the 1,2-phenylenediamine moieties alternate with each other as a result of the c-glide operation. In the column, an infinite, almost straight [Pt⋯Pt⋯Pt = 176.513 (11)°] platinum chain is formed with a short interatomic distance [Pt⋯Pt = 3.3475 (8) Å], suggesting weak metal–metal interactions. The infinite palladium chain of the isostructural Pd complex is straighter [Pd⋯Pd⋯Pd = 179.232 (7)°] than the platinum chain. The Pt⋯Pt distance in (I)cis-[PtCl2(NH3)2]     was slowly added an aqueous HCl solution  of 1,2-phenylenediamine , and then the solution was sealed in a screw-cap vial and was kept at room temperature for one week in the dark. Pale-brown needle-like crystals suitable for X-ray analysis were obtained (yield 52%). Elemental analysis found: C 19.26, H 2.23, N 7.30%; calculated for C6H8Cl2N2Pt: C 19.26, H 2.16, N 7.49%. Elemental analysis was carried out by the Laboratory of Organic Elemental Analysis, Department of Chemistry, Graduate School of Science, The University of Tokyo.Compound (I) al. 1972 as folloUiso(H) = 1.2Ueq. The maximum and minimum electron density peaks are located 0.80 and 0.74 Å, respectively, from atom Pt1.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017008477/wm5396sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989017008477/wm5396Isup2.hklStructure factors: contains datablock(s) I. DOI: 1055177CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II(ClO4)(mesoPYBP)](ClO4) {PYBP = 1,1′-bis[(pyridin-2-yl)methyl]-2,2′-bipiperidyl, C22H30N4}, was prepared and found to crystallize with two crystallographically independent complex salt moieties. The metal atoms of the cations adopt a pseudo-square-pyramidal coordination geometry, where the tetradentate aminopyridine ligands (PYBP) are wrapped around the Cu atoms in the equatorial plane.The title complex, [Cu II(ClO4)(mesoPYBP)](ClO4) {PYBP = 1,1′-bis[(pyridin-2-yl)methyl]-2,2′-bipiperidyl, C22H30N4}, was prepared and found to crystallize with two crystallographically independent complex salt moieties. The metal atoms of the cations adopt a pseudo-square-pyramidal coordination geometry, where the tetradentate aminopyridine ligands (PYBP) are wrapped around the Cu atoms in the equatorial plane. The Cu—O bonds involving an O atom of the coordinating perchlorate anion are approximately perpendicular to the plane. The two remaining non-coordinating perchlorate anions are involved in several C—H⋯O hydrogen bonds with the PYBP ligand and balance the total charge of the complex salt. The two crystallographically independent moieties are related to each other via a pseudo-translation along the a-axis direction. Exact translational symmetry is broken by (i) a difference in the conformation of one of the piperidine rings, featuring a chair conformation in one of the cations, and a sterically disfavored boat conformation in the other; and (ii) by modulation of the non-coordinating perchlorate anions.The title complex, [Cu Like some other CuII aminopyridine complexes , thus leading to a more favorable packing of the structure as a whole. As a result of the different conformations in the two complex cations, the Cu2—Nbp bonds [2.0226 (16) and 2.0078 (16) Å] differ by 0.015 Å, but the Cu2—Npy bonds [1.9901 (16) and 1.9890 (16) Å] are similar. In contrast, the piperidine rings of the other molecule (Cu1 moiety) are both in the more favorable chair conformation; the Cu1—Nbp distances [2.0349 (16) and 2.0365 (16) Å] are similar, but the Cu1—Npy distances [1.9808 (16) and 2.0309 (16) Å] differ. These Cu—N distances fall into the range of some other CuII aminopyridine complexes  complex, with Cu1—O and Cu2—O distances of 2.2038 (14) and 2.3438 (15) Å, respectively.As shown in Fig. 3A⋯O3ii; C17—H17B⋯O4ii; C28—H28B⋯O12iv); all of these hydrogen bonds are intermolecular, linking with the hydrogen atoms on the pyridine α-carbons of the adjacent Cu-mesoPYBP cations. The perchlorate close to the Cu1 moiety forms six hydrogen bonds with four adjacent complex cations (both Cu1 and Cu2), while that close to the Cu2 moiety only forms three hydrogen bonds with two adjacent complex cations (Cu2 only). This difference in hydrogen-bonding environments of the two outer-sphere perchlorates breaks the symmetry between them and between the cation moieties. All C⋯O distances of the C—H⋯O interactions (3.08–3.29 Å) are roughly equal to or shorter than the sum of van der Waals radii of the corresponding atoms (3.25 Å), indicating normal strength interactions.Details of hydrogen-bonding parameters are listed in Table 1mesoPYBP ligand involves two steps. A detailed synthetic procedure for -2,2′-bipiperidine-1,1′-diium dibromide (mesoBP·2HBr) via reductive hydrogenation of 2,2′-dipyridyl was reported by Herrmann et al. : 8.60 ; 8.24 ; 7.80 ; 7.73 ; 4.25 ; 3.55 ; 3.07 ; 2.84 ; 2.05 ; 1.78 ; 1.66 ; 1.51 . 13C NMR (CDCl3): 161.3; 149.0; 136.5; 122.5; 121.6; 63.8; 60.3; 54.2; 27.7; 24.9; 24.8. The mesoPYBP·xHBr was basified with excess NaOH in aqueous solution, and extracted with CH2Cl2. The CH2Cl2 solution was dried over MgSO4 and solvents were removed by rotary evaporation, giving mesoPYBP as a colorless oil .The synthesis of the  al. 2006 and Yang al. 2013. 1.81 g mesoPYBP ligand was dissolved in 2 mL MeCN. 0.74 g (2 mmol) Cu(ClO4)2·6H2O (MW = 370.54g mol−1) was dissolved in minimal MeCN. The solutions were combined and stirred for two days; any precipitate was removed by filtration and discarded. 1.22 g (85%) [CuII(mesoPYBP)(ClO4)](ClO4) was obtained as dark-blue crystals by slow evaporation of the MeCN solution. The crystals decomposed and became black within 15 minutes at 503 K (caution: heating perchlorate-containing compounds may lead to explosion). UV–Vis λmax: 625 nm (molar absorptivity: 0.59 L mol−1 cm−1). IR (in KBr pellet) νmax: 3071, 2958, 1610, 1444, 1081, 1025, 780 cm−1.Under ambient atmosphere, 0.70 g (2 mmol) 2, 1.00 Å for CH and 0.95 Å for aromatic CH bonds. Isotropic displacement parameters were set to 1.2 times of the equivalent isotropic displacement parameter of the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017009410/zl2706sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017009410/zl2706Isup2.hklStructure factors: contains datablock(s) I. DOI: 1558167CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: