The obtained structure was compared with the known hexanuclear silver(I) complex {[Ag(μ-L1Clpz)]3}2. The Ag···Ag distances in n[Ag(μ-L1Clpz)] are slightly shorter than twice Bondi’s van der Waals radius, indicating some Ag···Ag argentophilic interactions. Two Ag–N distances in n[Ag(μ-L1Clpz)] were found: 2.0760(13) and 2.0716(13) Å, and their N–Ag–N bond angles of 180.00(7)° and 179.83(5)° indicate that each silver(I) ion is coordinated by two pyrazolyl nitrogen atoms with an almost linear coordination. Every five pyrazoles point in the same direction to form a 1-D zig-zag structure. Some spectroscopic properties of n[Ag(μ-L1Clpz)] in the solid-state are different from those of {[Ag(μ-L1Clpz)]3}2 , presumably attributable to this zig-zag structure having longer but differently arranged intramolecular Ag···Ag interactions of 3.39171(17) Å. This result clearly demonstrates the different physicochemical properties in the solid-state between 1-D coordination polymer and metalacyclic trinuclear (hexanuclear) or tetranuclear silver(I) pyrazolate complexes.Coinage metal(I)···metal(I) interactions are widely of interest in fields such as supramolecular assembly and unique luminescent properties, etc. Only two types of polynuclear silver(I) pyrazolato complexes have been reported, however, and no detailed spectroscopic characterizations have been reported. An unexpected synthetic method yielded a polynuclear silver(I) complex  Ho. Ho3H3N2− (imidazolate) < Pyridine < Cb− (carbeniate) < Pz− for a given metal 3)2}](BF4) with Au···Ag interactions was reported (bzim− = 1-benzylimidazole anion) − as a silver(I) ion source. The dicyanoargentate(I) anion is very useful to build coordination polymers [n[Ag(μ-L1Clpz)]   pyrazolato complexes, it should be valuable to explore the different strategy of using electro-withdrawing substituents on the pyrazole ligand and  in ethyl acetate was carried out at room temperature as shown in nBu4N)[Ag(CN)2] at 2141 cm−1 disappeared and a new complex that did not contain any cyanide was generated. From a slow liquid-liquid diffusion method, we obtained a few single-crystals to reveal the structure (see 3/[Ag(CN)2]− based polynuclear structure with intermolecular Ag···Ag interactions was initially targeted, only the polynuclear silver(I) complex n[Ag(μ-L1Clpz)], identified as a coordination polymer was obtained ]3}2 or the other halogenated derivatives {[Ag(μ-L1Brpz)]3}2 and {[Ag(μ-L1Ipz)]3}2 (nBu4N)[Ag(CN)2] did not produce any powder precipitate. Indeed, after all of the solvent had slowly evaporated from the reaction mixture, powder XRD measurements were carried out on the resulting residues, which indicated that no new complexes were generated: only a physical mixture of the initial starting materials, i.e., the hexanuclear complexes {[Ag(L1Xpz)]3}2  and (nBu4N)[Ag(CN)2] were observed. We also tried to make polynuclear [Ag(μ-L1Xpz)]n  by the literature method by using aqueous NH3 [3}2  were obtained.The reaction of {[Ag(μ-L1Clpz)]ture see . The aboobtained ; the detIpz)]3}2 . The reaeous NH3 ,16, but n[Ag(μ-L1Clpz)], the perspective drawing of which is shown in n[Ag(μ-L1Clpz)] and packing diagram are drawn in Single-crystal X-ray structural analysis was performed on the polynuclear silver(I) material n[Ag(μ-L1Clpz)] exists as a coordination polymer with an intramolecular argentophilic distance of Ag1···Ag2, 3.39171(17) Å, which is slightly shorter than twice Bondi’s van der Waals radius (3.44 Å = 1.72 Å × 2)  structure is completely different from the reported one for [Ag(pz)]n which exhibits an intramolecular Ag···Ag distance of 3.3718(7) Å. The zig-zag chain in [Ag(pz)]n also interacts with neighboring zig-zag chains via inter-chain Ag···Ag distances of 3.2547(6) Å (n[Ag(μ-L1Clpz)] is also different from the original hexanuclear silver(I) complex {[Ag(μ-L1Clpz)]3}2, which features a dimeric trinuclear structure with three intermolecular argentophilic interactions of 3.1003(17), 3.1298(15), and 3.1051 Å to form an overall hexanuclear structure (3}2 did not interact with the neighboring layered one (3}2 complexes of 7.3531(18), 7.3881(17), and 7.5258(18) Å.This 547(6) Å . The zigtructure  20]. Th. Th[Ag(μtructure . The layered one . The lay1H-NMR spectrum of the obtained white powder n[Ag(μ-L1Clpz)] in CDCl3 was measured (3}2 (n[Ag(μ-L1Clpz)] is not stable upon dissolution, converting to form the known hexanuclear silver(I) complex {[Ag(μ-L1Clpz)]3}2. This observation is also supported by its solution-state UV-Vis spectrum in cyclohexane (3}2 (226 nm)  and {[Ag(μ-L1Clpz)]3}2 were carried out by solid-state spectroscopies.The measured  and the red ] and {[Ag(μ-L1Clpz)]3}2 complexes are reproduced in −1 (IR) and of 1507 and 1495 cm−1 (Raman) in n[Ag(μ-L1Clpz)] are almost the same as the C=N stretching vibrations of 1505 cm−1 (IR) and 1495 cm−1 (Raman) in {[Ag(μ-L1Clpz)]3}2. The C–Cl stretching vibrations could be observed in the far-IR region of 587 cm−1 (IR) and 575 cm−1 (Raman) in n[Ag(μ-L1Clpz)], which are slightly shifted from 579 cm−1 (IR) and 573 cm−1 (Raman) in {[Ag(μ-L1Clpz)]3}2 [−1 [−1 (Raman) in n[Ag(μ-L1Clpz)], which was the same energy at 511 cm−1 (Raman) in {[Ag(μ-L1Clpz)]3}2 [IR and Raman spectra of the lpz)]3}2 , respect]3}2 3}2 . These vn[Ag(μ-L1Clpz)] acquired as a Nujol suspension is shown in 3}2 [3}2 [n[Ag(μ-L1Clpz)] may be caused by polynuclear formation with intramolecular argentophilic interactions of Ag1···Ag2, 3.39171(17) Å as shown in The solid-state UV-Vis absorption spectrum of n in 3}2  for comp3}2 [3}2  was obvin[Ag(μ-L1Clpz)] in the solid-state was also somewhat different from that of the hexanuclear silver(I) analogue {[Ag(μ-L1Clpz)]3}2  was clearly shifted to higher energy around 314 nm, and a new broad peak around 490 nm was observed. The emission spectrum of lpz)]3}2  as shownn[Ag(μ-L1Clpz)] were recorded ] at 83 K exhibited an additional vibrational fine structure around 346 nm, which was observed only at 83 K. From this vibrational behavior, this higher energy emission may be from ligand-based phosphorescence. On the other hand, the lower energy emission band was attributed to metal-based phosphorescence arising from closed shell d10–d10 intramolecular Ag···Ag interactions  is very unique due to its polynuclear supramolecular structure. We are now in the process of probing the origin of this stimulating behavior by theoretical and more detailed physicochemical researches.The temperature-dependent photoluminescence spectra in recorded . The corractions ,32,33,34nBu4N)[Ag(CN)2] was obtained by the reaction of KAg(CN)2  and nBu4NBr  in 20 mL of H2O at room temperature for 2 hours to form a white powder of (nBu4N)[Ag(CN)2] . Hexanuclear silver(I) complexes ({[Ag(μ-L1Clpz)]3}2, {[Ag(μ-L1Brpz)]3}2, and {[Ag(μ-L1Ipz)]3}2) were obtained by the published methods   were dissolved in ethyl acetate (6 cm3) and the solution was stirred at room temperature for one week. A white solid was precipitated during this time. After this, the white solid was filtered and washed with a small amount of ethyl acetate. The obtained white powder as n[Ag(μ-L1Clpz)] was dried by vacuum pump.{[Ag(μ-L1Clpz)]Yield: 22.7 mg, 0.077 mmol, 42%.9H14AgClN2·1/4(H2O): C, 36.27; H, 4.90; N, 9.40. Found: C, 36.21; H, 4.57; N, 9.20. IR : 2971s(C-H), 2925s(C-H), 2862s(C-H), 1506m(C≡N), 1461m, 1400m, 1367m, 1361m, 1283m, 1145s, 1118s, 1092s, 1051s, 1035s, 804w. Far-IR: 685w, 587m(C-Cl), 561w, 540w, 501,476w, 437w, 411w, 389w, 371w, 349w, 276w, 237br. Raman : 2974s(C-H), 2913s(C-H), 2865s(C-H), 1507m(C≡N), 1495m(C≡N), 1461m, 1448m, 1430m, 1382m, 1369s, 1300m, 1283m, 1176w, 1145w, 1107m, 957w, 880m, 706w, 657m, 575m(C-Cl), 511m(Ag-N), 438w, 385w, 341w, 268m. 1H-NMR : δ 1.41 , δ 3.13 . UV-Vis ) 226 (39200). UV-Vis : 248, 280 (sh). Emission at 280 nm excitation wavelength : 307. Emission at 280 nm excitation wavelength : 299 K, 315, 490; 173 K, 314, 474, 614sh; 83 K, 317, 331, 346, 362, 379, 456sh, 473.Calcd for C3}2 (0.05 mmol) in 10mL of ethyl acetate was transferred to a 30 mL Erlenmeyer flask. On this solution, the solution containing (nBu4N)[Ag(CN)2] (0.1 mmol) dissolved in 10 mL of ethyl acetate was carefully layered and then the top was covered by parafilm. After a few weeks, some crystals were formed, which were suitable for single-crystal X-ray analysis.Liquid-liquid diffusion was applied to obtain crystals at room temperature. The {[Ag(μ-L1Clpz)]n[Ag(μ-L1Clpz)] are given in Crystal data and refinement parameters for the silver(I) pyrazolato coordination polymer θ value of 55.0°. A total of 744 oscillation images were collected. The highly redundant data sets were reduced using CrysAlisPro  as a coordination polymer by the reaction of {[Ag(μ-L1Clpz)]3}2 with (nBu4N)[Ag(CN)2]. This polynuclear silver(I) structure was compared with the known hexanuclear silver(I) structure {[Ag(μ-L1Clpz)]3}2. Two N–Ag–N bond angles of 180.00(7)° and 179.83(5)° in the silver(I) coordination polymer n[Ag(μ-L1Clpz)] indicate that each silver(I) ion is coordinated by two pyrazolyl nitrogen atoms with an almost linear coordination. Every five pyrazoles point in the same direction to form a 1-D zig-zag structure. This zig-zag structure is not stable in solution, but it converts to the original hexanuclear silver(I) complex {[Ag(μ-L1Clpz)]3}2. In the solid-state photoluminescence spectrum, a lower energy strong emission band at 473 nm at lower temperature is very unique and attributable to the differences in polynuclear structure between the two systems. Silver(I) complexes are not generally so emissive, even at lower temperature and thus this 1-D zig-zag polynuclear structure is particularly noteworthy in coinage metal(I) pyrazolate research. This n[Ag(μ-L1Clpz)] broadens a family of silver(I) coordination polymers [By using an unexpected synthetic method, we obtained a silver(I) pyrazolato complex polymers ,41,42,43polymers . Further


The formation of the 1,2-OH free product N3-RibAFU-OH and its methyl glycoside methyl ester, N3-RibAFU(Me)-OMe were monitored by RP-HPLC and found that either 50% TFA or 8 eqv. Amberlite IR-120 H+ resin in MeOH are optimal reagents for the effective deprotection. These conditions were then successfully applied for the synthesis of chimeric oligopeptide: -GG-X-GG- [X=RibAFU(ip)]. We found the established conditions to be effective and—at the same time—sufficiently mild to remove 1,2-O-isopropylidene protection and thus, it is proposed to be used in the synthesis of oligo- and polypeptides of complex sequence combination.Complementary to hydrophobic five membered ring β-amino acids (e.g. ACPC), β-sugar amino acids (β-SAAs) have found increasing application as hydrophilic building blocks of foldamers and α/β chimeric peptides. Fmoc-protected β-SAAs [e.g. Fmoc-RibAFU(ip)-OH] are indeed useful Lego elements, ready to use for SPPS. The removal of 1,2-OH isopropylidene protecting group increasing the hydrophilicity of such SAA is presented here. We first used NThe online version of this article (10.1007/s00726-020-02923-3) contains supplementary material, which is available to authorized users. Numerou, d-xylo , 2 and dCPC Fig. .Fig. 1SoThe stable protection of the hydroxyl groups of β-SAA is essential during the synthesis of oligo- and polypeptides. Afterwards, the removal of their protecting groups is of a special interest to enhance the hydrophilicity of the parent chimera peptides -OH (7) and Fmoc-RibAFU(ip)-OH (8) β-SAA have their 1,2-O-isopropylidene protecting groups originating from 1,2;5,6-di-O-isopropylidene-d-glucofuranose (5) 2·6H2O   Scheme . Note thO-isopropylidene removal from SAAs oligopeptides is known. Only 2,3-O-isopropylidene removal was reported, when cyclic dimer of protected 5-aminomethyl-3,4-dihydroxy-tetrahydrofuran-2-yl-acetic acid (H-SAA-OH) without 2,3-O-isopropylidene acetal was formed in aqueous TFA  was synthesized and the 2,3-O-isopropylidene protection was removed with aqueous HCl, following the peptide synthesis  free the furanoid 1,2-OHs to increase the hydrophilicity of the molecule and (2) form methyl glycoside to prevent furanoid ring opening. Consequently, we synthesized our model oligopeptide Ac-GG-X-GG-R  cannot occur.O-isopropylidene protection, the easily accessible intermediate N3-RibAFU(ip)-OH (7) (Scheme 3-RibAFU(ip)-NHMe (11)  was completed previously from d-Glc . Then, N3-RibAFU(ip)-OH (7) was obtained from 6 and in the final steps Fmoc-RibAFU(ip)-OH (8) product was achieved. We have shown that Fmoc-RibAFU(ip)-OH (8) is a suitable monomer for solid-phase peptide synthesis (SPPS), as both the 1,2-OHs and the amino function are protected -OH .The deprotection was carried out under strong acidic conditions Scheme  and the A: Compound 7 was dissolved in the mixture of TFA in DCM of 50%, 70% and 90% concentrations in parallels together with a small amount of H2O (2.5%) and TIS (2.5%) as scavengers. All reactions were carried out at room temperature. Analysis of RP-HPLC diagrams resulted in evaluable data of the reaction with varied concentration of TFA. Since component 9 has hydrophilic character during the deprotection reactions, instead of a conventional C-18 column, Hydro-RP LC column was used for RP-HPLC system. The hydrolysis of 7 (retention time 21.6 min) was almost completed and no intermediate was detected . The new signal at 4.3 min was indicated and the reaction mixture was processed to give white oil, however, attempts to isolate a pure product failed.In Route 9 such mixture of the anomers could be expected. This fact was indicated by ESI–MS exhibiting a peak at m/z 188.03029  of the unprotected carboxylic acid (9). Further studies with HILIC LC–MS (Hydrophilic Interaction Chromatography) corroborated the presence of 9 in the mixture. In these chromatograms, the signal at 2.13 min was assigned to the starting 7 based on the ESI–MS m/z 228.06094 [M−H]− . Furthermore, the signal at 10.46 min was attributed to the product 9 in accordance with ESI–MS m/z 188.03057 [M−H]− . This expected product (9) might be also highly unstable, as solely the related O-protected derivatives were described in the literature -NHMe (11) in TFA/DCM (50%) with TIS (2.5%) and H2O (2.5%) was stirred at room temperature for 1 h to be completed according to TLC and RP-HPLC . The signal of the expected product 12 appeared at about 2 min and indicated the mixture of products also in this case. Working up the reaction mixture gave a solid which could not be separated to give pure anomers. Formation of more products was attributed to the free OH-1 in 12—as in the case of 9. Additional reasons for the dispersity of the peak at 2 min was revealed by ESI–MS exhibiting the isotopic peak of an isobaric contaminant at m/z 203.822 beside of the peak of the mixture of 12 at m/z 203.07803 .Analogue reaction of NB: Compound 7 was dissolved in the solution of TFA/dried MeOH in parallel concentrations of 30%, 50% and 70%. The mixtures were kept for 18 h at room temperature then were processed to give oily products from each sample. Analysis with RP-HPLC revealed complex mixtures in all cases . Only the decreasing signal of the starting 7 was unambiguously assigned between 11.7 and 12 min, respectively, depending on the TFA concentration. Further components—intermediates and side products—were detected in all cases. The formation of the intermediate 14 in the reaction mixture of concentration of 30% TFA was identified . Attempted separation of the components failed from the oily mixtures, thus, Route B was not continued.In Route C: These reactions of the starting 7 were carried out under heterogeneous conditions: Amberlite IR-120 H+ resin was used as the acid component in dried MeOH. The mol ratio was in parallel reactions: 2, 4, 8 and 12 eqv. The mixtures were kept at 60 °C, 40 °C and at room temperature till to the reaction was complete. Analysis of RP-HPLC chromatograms revealed that 1,2-O-isopropylidene deprotection on 7 (11.9 min) occurred, parallel, with the formation of the methyl ester methyl furanoside (10) as final main product (9.0 min). Besides, two further peaks were detected at 6.8 min and at 15.3 min, respectively. These were reasonably assigned to the temporary intermediate methyl esters (14 and 15). The best results were obtained from the reaction with 8 eqv. Amberlite IR-120 H+ at 60 °C when almost complete transformation occurred in 360 min  and also intermediate 15   disappeared in the spectrum of the methyl glycoside product (10). At the same time, the spectrum of the latter exhibited the characteristic methyl signals of the methyl ester and the methyl O-glycoside at 3.84 and 3.42 ppm, respectively  is the main component and the β-anomer (3J couplings of H1-H2: 11.5 Hz) is the minor one. ESI–MS exhibited m/z [M+Na]+ 240.1 for C7H11N3O5 supporting the molecular composition of 10. The small quantity of the minor component did not allow the detailed analysis, however, the evident presence of 15 in the reaction mixture was supported by the mechanism of the complex reaction (Scheme Working up the mixture gave inseparable oil. The 3-RibAFU(ip)-NHMe (11) was carried out under the best conditions as in the case of 7: with Amberlite IR-120 H+ (8 eqv.)/dried MeOH at 60 °C and completed within 300 min. Analysis of RP-HPLC diagrams revealed that 1,2-O-isopropylidene deprotection on 11 (9.9 min) occurred, parallel, with the formation of the expected methyl furanoside (13) as final main product . Besides, a second product was observed at 3 min, probably, the 1,2-(OH)2 derivative (12). Working up the reaction mixture gave an inseparable oily product. The main component of the mixture was identified by ESI–MS which exhibited two strong peaks (m/z 217.09303 [M+H]+ and m/z 239.07487 [M+Na]+) corresponding to the structure of 13. Other two peaks were also measured (m/z 203.07725 [M′+H]+ and m/z 225.19593 [M′+ Na]+) which were attributed to the side product 12 in the mixture .Analogue reaction of NX-GG- model system was synthesized here with X=RibAFU(ip) and conditions to remove its 1,2-O-isopropylidene protecting group were probed. The synthesis of -GG-RibAFU(ip)-GG- was carried out with Fmoc-strategy introducing Fmoc-RibAFU(ip)-OH (8) on RAM-Tentagel® or 2-Cl-Trt-Cl resin (Scheme N-terminus of this model peptide were protected to avoid unwanted side-reactions (e.g. esterification). Peptide 18 was used for evaluation of peptide synthesis method and an intermediate of methyl glycoside formation. The deprotection of 1,2-O-isopropylidene moiety was carried out with the mixture of TFA/DCM/TIS/H2O which is in fact a common cleavage cocktail in SPPS. The 50% TFA condition was used to remove peptides from the resin and in parallel to complete the 1,2-O-isopropylidene deprotection (Scheme e). The removal of the 1,2-O-isopropylidene group gave successfully both pentapeptides with free 1,2-OHs . In the mixture of the crude product, 4:1/α:β anomeric ratio was observed at 11.29 min and 12.04 min, respectively  using two routes. The Amberlite IR-120 H+ (8 eqv.)/MeOH condition was executed on compound 18 with 1,2-O-isopropylidene protection (Route D) and on compound 17 with fully unprotected SAA (Route E). In both cases, the Ac-GG-RibAFU(Me)-GG-OMe (19) pentapeptide was observed. However, Route E was faster for methyl glycoside formation, with retention times of 4.75 min and 5.54 min presenting a 4:1/α:β anomeric ratio, respectively .Our ultimate goal was to apply the currently fine-tuned deprotection method for making various peptides and chimera sequences. The previously successfully applied -GG-n Scheme  by PyBOPn Scheme . The N-tn Scheme , step e.9, 12, 13) and peptides  having a short retention time due to their increased hydrophilicity. The analysis of these polar compounds was a challenge, as their interaction with C-18 stationary phases was insufficient, leading to a very difficult product isolation. Products  were characterized via HILIC LC–UV–MS -OH synthetic intermediate. To obtain the unprotected derivatives, two different conditions were studied successfully, namely (1) various TFA concentrations to form the free 1,2-OH product (9) and (2) various equivalents of Amberlite IR-120 H+ resin or TFA in MeOH forming methyl glycoside (10) to prevent the furanoid ring opening. In the first case, 50% TFA in DCM with TIS and H2O as scavengers was found to give the fully unprotected compound . Furthermore, 8 eqv. Amberlite IR-120 H+ resin in MeOH at 60 °C turned out to be optimum to furnish methyl glycoside methyl ester N3-RibAFU(Me)-OMe (10). These optimized methods, 50% TFA and H+ resin/MeOH, were applied successfully during the synthesis of α/β-chimera oligopeptide [-GG-RibAFU(ip)-GG-] to form the Ac-GG-RibAFU-GG-NH2 (16) without 1,2-O-isopropylidene protection and the methyl glycoside variant, namely the Ac-GG-RibAFU(Me)-GG-OMe (19). The described, fine-tuned conditions of deprotection were shown to be appropriate and sufficiently mild to remove 1,2-O-isopropylidene protection in oligo- and polypeptides of more complex amino acid sequences.A rather general 1,2-+ resin was 1.80 eq/L. Reactions were followed by RP-HPLC on a Phenomenex Jupiter C-18 column or Synergy™ 4 μm Hydro-RP 80 Å LC column with eluents 0.1% TFA in H2O (A) and 0.08% TFA, 95% acetonitrile/5% H2O (B), flow rate 1.0 ml/min and UV detection at 220 and 280 nm. Gradient were as follows: 0 min: 0% B, 30 min: 60% B, 32 min: 95% B, 33 min: 0% B, 45 min: 0% B and 45.1 min: 0% B for Phenomenex Jupiter C-18 column, and 0 min: 0% B, 30 min: 60% B, 33 min: 95% B, 39 min: 95% B, 40 min: 0% B, 45 min: 0% B and 45.2 min: 0% B for Synergy™ 4 μm Hydro-RP 80 Å LC column. MS spectra were performed with Bruker Esquire 3000 + tandem quadrupole mass spectrometer equipped with an electrospray ion source. FTIR spectra were recorded on a Bruker IFS 28 spectrometer by ATR technique. 1H NMR measurements were implemented with Bruker Avance 250 spectrometer in CDCl3 or DMSO-d6 at room temperature. Deuterated solvents were purchased from Eurisotop. Hydrophilic compounds were analyzed by HILIC LC–UV–MS approach. The measurements were executed on a column Waters Acquity BEH Amide UPLC  using 20 mM ammonium acetate (A) and 100% acetonitrile (B) with flow rate 250 µl/min, UV detection at 210 and 280 nm and 40 °C column temperature. Dionex 3000 UHPLC was coupled to a Q Exactive Focus orbitrap mass spectrometer . ESI–MS spectra were acquired in m/z 200–800 . MS/MS spectra were acquired using higher energy collision induced dissociation (HCD) at 20 eV. Gradients were as follows: 0 min: 90% B, 2 min: 90% B, 22 min: 40% B, 23 min: 40% B, 24 min: 90% B and 30 min: 90% B for peptides, and 0 min: 95% B, 2 min: 95% B, 22 min: 60% B, 23 min: 60% B, 24 min: 95% B and 30 min: 95% B for SAAs.Reagents, materials and solvents were obtained from Sigma-Aldrich, Merck, Reanal and VWR. Moisture-sensitive solvents were dried on molecular sieve (3 Å). The capacity of Amberlite IR-120 H7 was prepared based on previous results of our group , 2112 (N3), 1724 (C=O); ESI–MS: m/z calculated for C8H11N3O5 [M−H]− 228.06205, found 228.06207; 1H NMR  δ ppm 5.84 , 4.78 , 4.35 , 3.85 , 1.44 , 1.28 ; HILIC LC–UV–MS: 2.13 min, m/z calculated for C8H11N3O5 [M−H]− 228.06204, found 228.06094.Compound 8 was prepared based on previous results of our group , 3367 (NH), 1720 (C=O), 1692 (aromatic); ESI–MS: m/z calculated for C23H23NO7 [M+H]+ 426.2, found 426.2; 1H NMR  δ ppm 7.89 , 7.69 , 7.36 , 5.85 , 4.61 , 4.29 , 4.01 , 1.46 , 1.26 .Compound O-isopropylidene-3-azido-3-deoxy-α-d-ribofuranuronic acid  was dissolved in DCM (49 ml), then CH3NH2·HCl (120 mg), EDC·HCl (671 mg), DIEA (0.6 ml) and HOBt (235 mg) were added. The mixture was stirred at room temperature for 3 h (EtOAc-Hex 2:1). After the reaction was complete, DCM (25 ml) was added to the mixture and extracted with 2 N HCl (2 × 50 ml), then washed with H2O (1 × 50 ml), saturated NaHCO3 solution (1 × 50 ml) and H2O (1 × 50 ml). The organic phase was dried (Na2SO4), filtered and concentrated in vacuo. The residue was treated by hexane to afford the product as white solid . RP-HPLC: 9.9 min; ESI–MS: m/z calculated for C9H14N4O4 [M+H]+ 243.10933 and [M+Na]+ 265.09128, found 243.10859 and 265.09035, respectively; 1H NMR  δ ppm 6.43 , 5.84 , 4.71 , 4.48 , 3.65 , 2.86 , 1.57 , 1.37 .1,2-O-Isopropylidene-3-azido-3-deoxy-α-d-ribofuranuronic acid  was dissolved in 5 ml of different concentrations of TFA  in DCM  with TIS (2.5%) and H2O (2.5%). The mixtures were stirred at room temperature for 3 h and evaporated in vacuo. The residues were dissolved in 1,2-dimethoxyethane (5 ml) and concentrated to obtain white oils in both cases. RP-HPLC: 4.3 min; ESI–MS: m/z calculated for C5H7N3O5 [M−H]− 188.03075, found 188.03029; HILIC LC–UV–MS: 10.46 min, m/z calculated for C5H7N3O5 [M−H]− 188.03074, found 188.03057.(A) 1,2-O-Isopropylidene-3-azido-3-deoxy-α-d-ribofuranuronic acid  was added into 2.5 ml mixtures of TFA  in DCM , TIS (2.5%) and H2O (2.5%). Reactions were stirred at room temperature. Fractions of reaction mixtures were concentrated by air blowing, diluted in water then measured by RP-HPLC .(B) 1,2-O-Isopropylidene-3-azido-3-deoxy-α-d-ribofuranuronic acid  was dissolved in dried MeOH (2 ml) and Amberlite IR-120 H+ resin  were added. The mixtures were heated to 60 °C for 6 h. The reactions were followed by RP-HPLC . The mixtures were filtered and washed with MeOH. Filtrates were evaporated in vacuo to obtain oil products. The mixture of products containing mainly the final product 10 (~ 90%) was characterized. RP-HPLC: 9.0 min; ESI–MS: m/z calculated for C7H11N3O5 [M+Na]+ 240.1, found 240.1; 1H NMR  δ ppm 4.93 , 4.58 , 4.36 , 4.16 , 3.83 , 3.42 .(A) 1,2-O-Isopropylidene-3-azido-3-deoxy-α-d-ribofuranuronic acid  was dissolved in dried MeOH (2 ml) and Amberlite IR-120 H+ resin  was added to the solution. The reaction mixture was stirred at different temperatures . Fractions of reaction mixtures were neutralized (NaHCO3), centrifuged, diluted in MeOH then measured by RP-HPLC .(B) 1,2-O-Isopropylidene-3-azido-3-deoxy-α-d-ribofuranuronic acid  was added into 2 ml mixtures of TFA  and dried MeOH . Reactions were stirred at room temperature overnight and evaporated in vacuo to obtain oil product. Mixtures were measured by RP-HPLC .(C) 1,2-11  was dissolved in the mixture (2 ml) of TFA (50%), DCM (45%), TIS (2.5%) and H2O (2.5%) and stirred at room temperature for 1 h. The reaction was monitored by TLC (EtOAc-Hex 2:1) and RP-HPLC . The solvent was removed in vacuo and the residue was treated with cold Et2O to precipitate the solid product. ESI–MS: m/z calculated for C6H10N4O4 [M+H]+ 203.07803, found 203.07722.Compound 11  was dissolved in dried MeOH (1.5 ml) and Amberlite IR-120 H+ resin  was added to the solution. The reaction was stirred at 60 °C for 6 h, monitored by TLC (EtOAc-Hex 2:1) and RP-HPLC. The mixture was filtered and washed with MeOH. The filtrate was concentrated in vacuo to achieve the oil product. ESI–MS: m/z calculated for C7H12N4O4 [M+Na]+ 239.07563 and [M+H]+ 217.09368, found [M+Na]+ 239.07487 and [M+H]+ 217.09303, respectively .Compound ® or 2-Cl-Trt-Cl resin with the standard methodology using Fmoc-strategy. Resins were swollen in DCM. Coupling of Fmoc-Gly-Gly-OH to resins was implemented in two different methods. For RAM-Tentagel® resin, the first step was the removal of Fmoc-group conducted with 2% piperidine and 2% DBU in DMF (10 + 20 min). The coupling was accomplished using Fmoc-Gly-Gly-OH  dissolved in DMF and PyBOP (3 eqv.)/DIEA (6 eqv.) added to the solution. In the case of 2-Cl-Trt-Cl resin, the coupling was made with Fmoc-Gly-Gly-OH  which was dissolved in DMF and DIEA (3.75 eqv.) was added to the solution. Afterwards, amino acids were coupled to resins using reagent pairs PyBOP/DIEA in DMF. Coupling of amino acids lasted for 1 h, whereas that of Fmoc-RibAFU(ip)-OH finished in 3 h. After coupling, resins were washed with 3 × DMF, 3 × DCM, 2 × MeOH, 1 × Et2O and dried in vacuo. The capacity of the resin was determined by spectrometric measurement of the amount of Fmoc chromophore (Fmoc-piperidine adduct) released upon treatment of the resin with 50% piperidine in DMF (Chan and White 2O:DIEA:DMF (1:1.2:3) for 30 min.SPPS of the model peptide was executed manually on RAM-Tentagel® resin (50 mg) with TFA (50%), DCM (45%), TIS (2.5%) and H2O (2.5%) for 3 h. The resin was washed with 3 × DCM and 3 × MeOH, then solvent was removed in vacuo. The residue was treated with cold Et2O to precipitate white solid product (4.9 mg). HILIC LC–UV–MS: 11.29 min and 12.04 min, m/z calculated for C15H24N6O9 [M+H]+ 433.1683 and [M+Na]+ 455.1502, found 433.1676 and 455.1493, respectively.The peptide was cleaved from RAM-Tentagel2O (2.5%) for 3 h. The resin was washed with 3 × DCM and 3 × MeOH, then solvent was removed in vacuo. By treating the residue with cold Et2O, the white solid product was precipitated (14.1 mg). HILIC LC–UV–MS: 12.68 min and 12.94 min, m/z calculated for C15H23N5O10 [M+H]+ 434.15232, [M+Na]+ 456.13426 and [M+H–H2O]+ 416.14176, found 434.15109, 456.413304 and 416.14060, respectively.The peptide was cleaved from 2-Cl-Trt-Cl resin (150 mg) by a mixture of TFA (50%), DCM (45%), TIS (2.5%) and H2O. The solvent was removed in vacuo. The residue was precipitated in cold Et2O to obtain white solid product . HILIC LC–UV–MS: 9.91 min, m/z calculated for C18H27N5O10 [M+H]+ 474.18362, found 474.18276.The peptide was cleaved from 2-Cl-Trt-Cl resin (150 mg) with AcOH:MeOH:DCM (1:1:8) for 3 h. The resin was washed with 3 × DCM, 3 × iPrOH and 1 × Et17 (13 mg) was dissolved in dried MeOH with Amberlite IR-120 H+ (8 eqv.). The mixture was stirred at 60 °C for 3 h, filtered and washed with MeOH. The filtrate was concentrated in vacuo. The product as white solid was precipitated with cold Et2O (7.8 mg). HILIC LC–UV–MS: 4.75 min and 5.54 min, m/z calculated for C17H27N5O10 [M+H]+ 462.18362 and [M+H–CH3OH]+ 430.15741, found 462.18214 and 430.15641, respectively.(A) Peptide 18 (10 mg) was dissolved in dried MeOH with Amberlite IR-120 H+ (8 eqv.). The mixture was stirred at 60 °C for 6 h, filtered and washed with MeOH. The filtrate was concentrated in vacuo and the residue was treated in cold Et2O to obtain white solid product (6.7 mg).(B) Peptide Supplementary material 1 (PDF 2462 kb)Below is the link to the electronic supplementary material.


Helicobacter pylori colonises the stomach of approximately 50% of the global population. Cytotoxin-associated gene A protein (CagA) is one of the important virulent factors responsible for the increased inflammation and increases the risk of developing peptic ulcers and gastric carcinoma. The cytokine interleukin-6 (IL-6) has particularly important roles in the malignant transformation of gastric and intestinal epithelial cells as it is upregulated in H. pylori-infected gastric mucosa. In this study, we investigated the underlying mechanisms of CagA-induced IL-6 up-regulation during H. pylori infection. AGS cells, a human gastric adenocarcinoma cell line, lacking eEF1A1 were infected with CagA+H. pylori (NCTC11637), CagA−H. pylori (NCTC11637ΔcagA), or transduced by Ad-cagA/Ad-GFP. The expression and production of IL-6 were measured by quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The interactions among CagA, eukaryotic translation elongation factor 1-alpha 1 (eEF1A1), protein kinase Cδ (PKCδ), and signal transducer and activator of transcription 3 (STAT3) were determined by western blot or co-immunoprecipitation.H. pylori infection, CagA-M (residues 256‒871aa) was found to interact with eEF1A1-I (residues 1‒240aa). NCTC11637 increased the expression of IL-6 in AGS cells compared with NCTC11637ΔcagA whereas knockdown of eEF1A1 in AGS cells completely abrogated these effects. Moreover, the CagA-eEF1A1 complex promoted the expression of IL-6 in AGS cells. CagA and eEF1A1 cooperated to mediate the expression of IL-6 by affecting the activity of p-STATS727 in the nucleus. Further, CagA-eEF1A1 affected the activity of STAT3 by recruiting PKCδ. However, blocking PKCδ inhibited the phosphorylation of STAT3S727 and induction of IL-6 by CagA.During S727 in the nucleus. These findings provide new insights into the function of CagA-eEF1A1 interaction in gastric adenocarcinoma.CagA promotes the expression of IL-6 in AGS cells by recruiting PKCδ through eEF1A1 in the cytoplasm to increase the phosphorylation of STAT3 Helicobacter pylori infection and subsequent inflammation  for 15 min on ice. Nonidet P-40 was then added to a final concentration of 0.1%, and the samples were incubated for less than 5 min. The mixture was then centrifuged at 6000×g for 5 min at 4 °C. The supernatant was collected as the cytosolic extract and stored at −80 °C. The pellets were washed twice with hypotonic buffer A and resuspended again in hypertonic buffer C  for 1 h on ice and then centrifuged at 17,000×g for 15 min at 4 °C. The supernatant containing nuclear proteins was collected and stored at −80 °C. The protein concentrations of the cytosolic and nuclear extracts were determined using the BCA protein assay reagent.After Equivalent amounts of proteins (10–30 µg) were subjected to electrophoresis on 8% or 12% SDS–polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked in 5% fat-free dry milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature. The membranes were then incubated with primary antibodies in 3% fat-free dry milk in PBS overnight at 4 °C. Membranes were washed in PBST, followed by incubation with 1:2000 or 1:5000 dilutions of the corresponding HRP-conjugated secondary antibodies for 1.5 h and again washed with PBST. Immunoreactions were visualised with an ECL chemiluminescent detection kit (Intron).eEF1A1 and pcDNA3.1-Flag-cagA expression plasmids were constructed by standard molecular biology techniques. The optimized cagA gene fragment was inserted into an adenoviral vector  to construct the Ad-cagA adenovirus expressing CagA protein and control Ad-GFP virus. AGS-C and AGS-sheEF1A1 cells were transduced with Ad-cagA or Ad-GFP viruses (MOI = 30), respectively.All enzymes used for cloning procedures were purchased from New England Biolabs . The pcDNA3.1-Myc-S727 (1:1000), anti-MYC (1:1000), anti-FLAG (1:1000), anti-lamin B1 (1:1000), anti-CagA (1:200), anti-eEF1A1 (1:5000), and anti-GAPDH (1:10000).Briefly, cells were transfected with plasmids or infected as described above. Harvested cells were lysed on ice with lysis buffer. The lysates were incubated with primary antibodies and protein A/G Plus-Agarose beads overnight at 4 °C. The beads were washed three times with lysis buffer, and western blot analysis was performed, as described above. The following primary antibodies were used: anti-PKCδ (1:1000), anti-p-STAT3Taq Kit Takara) according to manufacturer’s instructions. RT-PCR was performed using the Real-Time PCR System (Applied Biosystems) following standard procedures. PCR conditions were 40 cycles at 95 °C for 30 s, 60 °C for 60 s, and 72 °C for 30 s. The primers used for RT-PCR are as follows: eEF1A1 forward, 5′-TCTGGTTGGAATGGTGACAA -3′ and reverse, 5′-ACGAGTTGGTGGTA GGATGC -3′; IL-6 forward, 5′-AGTGAGGAACAAGCCAGAGC -3′ and reverse, 5′-GAGGTG CCCATGCTACATTT -3′; β-actin forward, 5′-AGCCTCGCCTTTGCCGA-3′, and reverse, 5′-CTGGTGCCTGGGGCG-3 -3′. Amplified products were resolved by 2‒3% agarose gel electrophoresis, stained with SYBR Premix Ex Taq Kit (Takara), and visualised with the imagequant TM LAS 4000.Total RNA was isolated from AGS cells using Trizol and reverse transcribed into complementary DNA using OligodT primers and random hexamer primers  according to the manufacturer’s instructions.Supernatants were harvested and centrifuged at 1700×eEF1A1-RNA shRNA plasmid  or CON077 (Hu6-MCS-Ubiquitin-EGFP-IRES-puromycin) for 48 h, and then puromycin was added to the cells at a concentration of 500 ng/mL to screen the transfected cells. The stably transfected STAT3-knockdown cells were transfected with the LV-STAT3-RNA shRNA plasmid (target sequence: 5′-GCAAAGAATCACATGCCACTT-3′) and then cultured with puromycin (500 ng/mL).AGS cells were transfected with the LV-t test or ANOVA was used for statistical analyses with the GraphPad Prism 7.0 software . Differences between groups were considered significant when the P-value was < 0.05.Data are represented as the mean ± SD, and Student’s Additional file 1: Supplementary Fig. S1. CagA promotes increased NF-κB-p65 activity in the nucleus. AGS-C and AGS-sheEF1A1 cells were infected with NCTC11637 or NCTC11637ΔcagA bacterial solutions (MOI = 100). Precipitates were analysed by WB to detect NF-κB-p50 and NF-κB-p65. CagA cytotoxin-associated gene A, NF-κB-p50 nuclear factor kappa B-p50, NF-κB-p65 nuclear factor kappa B-p65, MOI multiplicity of infection, CON control, WB western blot.


The structures of four phosphorescent monoiridium and chloro-, cyanato- or oxamidato-bridged diiridium complexes with a potential for OLED applications are described. N)-9H-fluoren-3-yl-κC3]iridium pentane monosolvate, [Ir(C31H38NO)3]·C5H12, (I), di-μ2-chlorido-bis{bisiridium} pentane 0.3-solvate, [Ir2(C30H35FN)4Cl2]·0.3C5H12, (II), di-μ2-cyanato-bis{bisiridium} pentane monosolvate, [Ir2(C31H38NO)4(NCO)2(NCO)2]·C5H12, (III), and {μ-N,N′-bisoxamidato}bispyridin-2-yl]phenyl-κ2C1,N′}iridium)–chlorobenzene–pentane (1/2.3/0.4), [Ir2(C20H19N)4(C18H6F12N2O2)]·2.3C6H5Cl·0.4C5H12, (IV), synthesized in the quest for organic light-emitting devices, were determined. The bis-μ2-chloro and bis-μ2-cyanato complexes have ΔΔ and ΛΛ configurations of the distorted octahedral Ir centres in racemic crystals, whereas the oxamido complex has a centrosymmetric (meso) structure with the ΔΛ configuration. The bridging oxamido moiety has a nearly planar anti geometry. All structures show substantial disorder of both host molecules and solvents of crystallization.The crystal structures of tris[9,9-dihexyl-2-(5-methoxypyridin-2-yl-κ The Ir a) and 4-fluorinated (IIb) pyridyl rings   with the crystallographic molecular symmetry 222. Notably, the analogue of (II)c) also crystallized in a triclinic structure, with the lattice parameters and host molecules’ packing broadly similar to those of (II)et al., 2012c), only approximately conforms to C2 symmetry: the Ir2Cl2 system is not planar, as in (IIa) and (IIb), but folded by 7.7 (1)° (II)c) along the Cl⋯Cl vector. In (II)trans to one another and both C atoms trans to bridging chloro ligands. The mean bond lengths Ir—Cl = 2.507 (2), Ir—N = 2.036 (5) and Ir—C = 1.998 (6) Å are not substantially different from those in (IIa)  or (IIb) , although the Ir1⋯Ir2 distance in (II)a) or (IIb) . Both Ir centres in the molecule of (II)n-hexyl chains of the ligands show complicated disorder  occupancy.Complex (II)er Fig. 4, being sa) and (IIb), the Py/Py angles are 9.4 (3) and 6.3 (4)°, with parallel slips of 2.94 and 2.88 Å, respectively. In (IIc), the two Py/Py pairs are even more unequal than in (II)Near-parallel alignment of pyridyl rings coordinated to different Ir atoms  and IIIb [3.425 (1) Å] and ca 0.3 Å shorter than in chloro-bridged complexes. Similarly to (II)c), in each independent molecule one pair of pyridyl rings shows substantial π–π stacking , the other only a fringe overlap . In this structure, the n-hexyl chains also show extensive disorder er Fig. 6, which cmonoatomic bridges, both metal centres have to adopt the same chirality: the meso diastereomer with opposite (ΔΛ) configurations of the two iridium centres would have sterically impossible short intramolecular contacts between the cyclometalating ligands. With longer bridges, however, both the ΔΔ/ΛΛ and meso isomers are sterically possible – and in some cases have been isolated and structurally characterized trans positions to each other. The bridging oxamido ligand adopts the anti geometry, its central C2N2O2 moiety is almost planar, the 3,5-trifluoromethylated arene ring is inclined to its plane by 47.3 (1)° and the Ir atom deviates from it by 0.334 (4) Å, which amounts to an 11.2 (1)° fold of the chelate ring along the O1⋯N11 vector . In both independent cyclometalating ligands, the mesityl substituent is almost perpendicular to the pyridine ring [dihedral angles 85.9 (1) and 81.5 (1)°]. The Ir1—N1i bond of 2.182 (2) Å is substantially longer than in the analogue with a 4-tert-butylphenyl substituent at this N atom . The CF3 groups and the solvent of crystallization are disordered pyridine-C2,N′]-bis(μ-oxamidato)diiridium (IVa) with an unsubstituted oxamidato bridge, and its derivative with an N,N′-bis(4-tert-butylphenyl)-substituted bridge (IVb). As in (IV)anti-configuration of the bridge: (IVb) certainly, (IVa) most probably, although in the latter the bridge is disordered, as in (μ2-oxamidato)tetrakis(5-methyl-2-(pyridin-2-yl)phenyl)diiridium (V), reported by Graf et al. (2013b) and (V), like (IV)meso (ΔΛ) diastereomers where the Ir centres are related by a crystallographic inversion centre (IVb) or mirror plane (V), whereas molecule (IVa) lies on a crystallographic twofold axis in the centrosymmetric space group Pbcm, i.e. the crystal is a racemate of ΔΔ and ΛΛ diastereomers.Recently, we prepared and characterized two close analogues of (IV) al. 2013. It is nn-hexyl substituents, with the remaining gaps filled by pentane solvent molecules. There are few intermolecular contacts between aromatic moieties, and those of the edge-to-edge kind; no π–π stacking or intercalation exists. This promises better solubility and compatibility with the host polymer and, indeed, can explain the relatively high efficiency of these complexes in optoelectronic devices –(III) is defined by the flexible and extensively disordered et al., 2016a′), NIFKEU (IIa), NIFKAQ (IIb) and NIFJUJ (IIc), discussed above. A search for similar bis-cyanato-bridged complexes returned structures NIFJOD (IIIa) and NIFJIX (IIIb), also discussed above. No Ir(μ2-X)Ir analogues with other X ligands were found. A search for anti-oxamido-bridged diiridium complexes with two chelating phenylpyridyl moieties (with any substituents) at each Ir atom, returned three structures discussed above, viz. SAWDIG (IVa), SAWDEC (IVb) and GIRMIF (V). The only mono-iridium complexes cyclometalated by three fluorenyl-pyridino derivatives, are NIFJET  and tris(2-(pyridin-2-yl)spiro-3-yl)-iridium, SAWRIU 3 present in the CSD, KAVJOH is the trigonal polymorph  at 100 and 193 K, respectively, whereas KAVJOH02 3 or IrCl3, respectively, (III)i.e. di-chloro-bridged complex) with Bu4N+NCO−. All three complexes were recrystallized from the mixed solvents chlorobenzene/pentane.Complexes (I)et al., 2017N,N′-bisoxamide  in methanol (6 ml) and the corresponding dichloro-bridged dimer  to give (IV)m/z 1984.2 . Calculated for C98H78F12Ir2N6O2m/z 1984.53.Complex (IV)Crystal data, data collection and structure refinement details are summarized in Table 1^C37, ligand N1^C1 except the pyridyl ring and the methyl atom C67, and n-hexyl substituents (except atoms C75 and C76) in the ligand N2^C19 are disordered equally between two positions (one of them labelled B). In the pentane solvent molecule, all atoms except C(1S) are also disordered between two positions .In structure (I)n-hexyl chains attached to it (except the C82 atom), chains C93—C94—C95—C96 and C105—C106—C107—C108 (with their respective H atoms) are disordered over two sets of sites with occupancies 2/3 and 1/3. The butyl chain at C110 is disordered between positions C111—C112—C113—C114 and C211—C212—C213—C214, the butyl chain at C116 is disordered between positions C117—C118—C119—C120 and C217—C218—C219—C220 (with respective H atoms); both in a 0.6:0.4 ratio, respectively. The ethyl group at C100 is disordered between positions C101—C102 and C201—C202 in a 0.75:0.25 ratio. All minor positions are labelled B. The pentane molecule of crystallization, C1S—C2S—C3S—C4S—C5S (with respective H atoms) shares the site with disordered n-hexyl groups and was refined with a 0.3 occupancy.In structure (II)A—C10B—C10C—C10D, with occupancies 2/3 and 1/3, respectively, in both cases. A methylene group is disordered between positions C80′ and C80B with occupancies 0.6 and 0.4 and a dimethylene fragment is disordered between positions C204—C205 and C20B—C20C with occupancies 0.7 and 0.3, respectively. The propyl chain at C211 is disordered between positions C212—C213—C214 and C21B—C21C—C21D, and the butyl chain at C216 is disordered between positions C217—C218—C219—C220 and C21E—C21F—C21G—C21H with equal (0.5) occupancies in both cases. The structure contains enclosed solvent-accessible voids of 374 Å3 (four per unit cell), occupied by disordered solvent, which could not be refined at atomic resolution and was masked using the OLEX2 SMTBX solvent-masking procedure based on Rees et al. (2005In structure (III) al. 2005. The difIn structures (I)–(III), all H atoms were permitted to ride in geometrically idealized positions with C—H = 0.95, 0.99 and 0.98 Å for aromatic, methylene and methyl C atoms, respectively.Uiso for all H atoms of each group), except the C48H3 group, which was treated as ideally disordered between two opposite orientations. For the latter and aromatic H atoms, Uiso(H) = 1.2Ueq(C). The C31F3 group is disordered (by rotation about the C—C bond) between orientations A and B with occupancies of 0.774 (5) and 0.226 (5), respectively, while the C30F3 group is disordered by a similar rotation and tilt of the C—C bond, between orientations A and B with occupancies of 0.586 (15) and 0.414 (15), respectively. The chlorobenzene molecule in a general position has the chlorine atom disordered equally between positions Cl1 and Cl2. The void around the inversion centre  with the solvent-accessible volume of 204 Å3 is shared by ca 0.15 of a chlorobenzene and 0.20 of a pentane molecule, refined at atomic resolution; the occupancies are in agreement with the integral electron density of 33.4 e per void, as estimated by OLEX2 SMTBX. The benzene ring has crystallographic inversion symmetry: the Cl atom is disordered between two positions related by this inversion. The central pentane atom C61 lies at the inversion centre, the adjacent atom is disordered equally between two positions with the terminal atom ordered. The pentane H atoms were not located. A strong peak of residual electron density (4.2 e Å−3) near the Ir1 atom can be interpreted as an alternative position, Ir1, of this atom, due to a whole-molecule disordered by a 12° rotation around its  inversion centre. Refinement of the occupancies of Ir1 and Ir2  converged at 0.9817 (6) and 0.0183 (6), respectively, with a Ir1⋯Ir2 distance of 0.62 Å.In structure (IV)10.1107/S2056989020001784/hb7885sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, global. DOI: 10.1107/S2056989020001784/hb7885Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020001784/hb7885Isup6.cdxSupporting information file. DOI: 10.1107/S2056989020001784/hb7885IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989020001784/hb7885IIsup7.cdxSupporting information file. DOI: 10.1107/S2056989020001784/hb7885IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989020001784/hb7885IIIsup8.cdxSupporting information file. DOI: 10.1107/S2056989020001784/hb7885IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989020001784/hb7885IVsup9.cdxSupporting information file. DOI: 1982629, 1982628, 1982627, 1982626CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Bnz⋯ODiazp and C—HProprg⋯ODiazp  hydrogen bonds link the molecules into two-dimensional networks parallel to the bc plane, enclosing The title compound consists of pyrrole and benzodiazepine units linked to a propargyl moiety, where the pyrrole and diazepine rings adopt half-chair and boat conformations, respectively. In the crystal, weak C—H 15H14N2O2, consists of pyrrole and benzodiazepine units linked to a propargyl moiety, where the pyrrole and diazepine rings adopt half-chair and boat conformations, respectively. The absolute configuration was assigned on the the basis of l-proline, which was used in the synthesis of benzodiazepine. In the crystal, weak C—HBnz⋯ODiazp and C—HProprg⋯ODiazp  hydrogen bonds link the molecules into two-dimensional networks parallel to the bc plane, enclosing R44(28) ring motifs, with the networks forming oblique stacks along the a-axis direction. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (49.8%), H⋯C/C⋯H (25.7%) and H⋯O/O⋯H (20.1%) interactions. Hydrogen bonding and van der Waals interactions are the dominant interactions in the crystal packing. Computational chemistry indicates that in the crystal, C—H⋯O hydrogen-bond energies are 38.8 (for C—HBnz⋯ODiazp) and 27.1 (for C—HProprg⋯ODiazp) kJ mol−1. 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 HOMO–LUMO behaviour was elucidated to determine the energy gap.The title compound, C For their anticancer activity, see: Bose et al.  as catalyst and potassium carbonate as base  Å and θ = 94.4 (4)°] while the seven-membered diazepine ring (N1/N2/C1/C6–C9) adopts a boat conformation . In the propargyl moiety, the N2—C13—C14 and C13—C14—C15 bond angles are 112.66 (17)° and 177.4 (3)°, respectively.The title compound, (I)Bnz⋯ODiazp and C—HProprg⋯ODiazp  hydrogen bonds  analysis ; the pair of spikes have tips at de + di = 2.80 Å. The thin and thick pairs of scattered points of wings in the fingerprint plot delineated into H⋯O/O⋯H contacts  have a symmetrical distribution of points with the edges at de + di = 2.42 and 2.44 Å, respectively. The C⋯C contacts  have a pliers-shaped distribution of points with the tips at de + di = 3.47 Å. Finally, the H ⋯ N/N⋯H interactions (1.8%) are reflected in Fig. 6f as thick wings with the tips at de + di = 3.04 Å. Selected contacts are listed in Table 2The overall two-dimensional fingerprint plot, Fig. 6ts Fig. 6d, 20.1%ts Fig. 6e, 1.8% dnorm plotted onto the surface are shown for the H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions in Fig. 7a–c, respectively.The Hirshfeld surface representations with the function et al., 2015The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions suggest that van der Waals interactions and hydrogen bonding play the major roles in the crystal packing  is the sum of electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) energies  were calculated as −13.2 (Eele), −3.8 (Epol), −45.1 (Edis), 27.8 (Erep) and −38.8 (Etot) for C2—H2⋯O2 and −10.7 (Eele), −4.0 (Epol), −25.8 (Edis), 15.7 (Erep) and −27.1 (Etot) for C13—H13A⋯O1.The intermolecular interaction energies were calculated using the CE–B3LYP/6–31G energy model in via density functional theory (DFT) using standard B3LYP functional and 6–311 G basis-set calculations . The significance of η and σ is to evaluate both the reactivity and stability. The electron transition from the HOMO to the LUMO energy level is shown in Fig. 8c]benzodiazepine-5,11-dione ring. The energy band gap [ΔE = ELUMO − EHOMO] of the molecule is 3.4829 eV, and the frontier molecular orbital energies, EHOMO and ELUMO are −4.0030 and −0.5203 eV, respectively.The optimized structure of the title compound in the gas phase was generated theoretically viz. 10-allyl-2,3-dihydro-1H-pyrrolobenzodiazepine-5,11-dione -2,3,5,10,11,11a-hexahydro-1H-pyrrolobenzodiazepine-3,11-dione , propargyl bromide (2.15 mmol) and potassium carbonate (4.3 mmol) along with a catalytic amount of tetra-n-butyl ammonium bromide were stirred in N,N-dimethylformamide (20 ml) for 72 h. The solid material was removed by filtration and the solvent evaporated under vacuum. The residue was separated by chromatography on silica gel with an n-hexane–ethyl acetate (1:9) solvent system. The title compound was obtained as colourless crystals in 70% yield upon evaporation of the solvent.The synthesis of pyrrolobenzodiazepine is a simple condensation of isatoic anhydride on Uiso(H) = 1.Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989020002698/lh5947sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020002698/lh5947Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020002698/lh5947Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020002698/lh5947Isup4.cmlSupporting information file. DOI: 1986475CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atom in the title complex is octahedrally coordinated within an N2O4 donor set defined by two N-atom donors of the 1,10-phenanthroline ligand, sulfate-O and three aqua-O atoms, the latter occupying an octahedral face. In the crystal, supramolecular layers parallel to (110) are sustained by aqua-O—H⋯O(sulfate) hydrogen bonding.The Co II atom in the title complex, [Co(SO4)(C12H8N2)(H2O)3] (or C12H14CoN2O7S), is octahedrally coordinated within a cis-N2O4 donor set defined by the chelating N-donors of the 1,10-phenanthroline ligand, sulfate-O and three aqua-O atoms, the latter occupying an octahedral face. In the crystal, supramolecular layers lying parallel to (110) are sustained by aqua-O—H⋯O(sulfate) hydrogen bonding. The layers stack along the c-axis direction with the closest directional interaction between them being a weak phenanthroline-C—H⋯O(sulfate) contact. There are four significant types of contact contributing to the calculated Hirshfeld surface: at 44.5%, the major contribution comes from O—H⋯O contacts followed by H⋯H (28.6%), H⋯C/C⋯H (19.5%) and C⋯C (5.7%) contacts. The dominance of the electrostatic potential force in the molecular packing is also evident in the calculated energy frameworks. The title complex is isostructural with its manganese, zinc and cadmium containing analogues and isomeric with its mer-triaqua analogue.The Co The Co—N bond lengths are equal within experimental error but the Co—O(aqua) bonds span an experimentally distinct range, Table 1trans to a nitrogen atom suggests the differences in bond lengths are due to the considerable hydrogen bonding operating in the crystal. Indeed, there is an intramolecular aqua-O1W—H⋯O3(sulfate) hydrogen bond, Table 23 residue is delocalized over the three non-coordinating S—O bonds.The molecule of (I)a). The aqua molecule forming the intramolecular O1W—H⋯O3 hydrogen bond forms a second hydrogen bond to the coordinated O1 atom of a symmetry-related molecule, and the O2W aqua ligand of this molecule connects to the O3 atom of the original molecule, leading to the formation of a non-symmetric eight-membered {⋯HOH⋯O⋯HOCoO} synthon. The second hydrogen atom of the O2W ligand forms a connection to a sulfate-O4 atom, which is also hydrogen bonded to an O3W molecule, which forms an additional link to a symmetry related sulfate-O2 atom with the result a {⋯HOH⋯OSO⋯HOH⋯O} non-symmetric ten-membered synthon is formed. Two additional eight-membered synthons, {HOCoOH⋯OSO}, are formed as a result of the hydrogen-bonding scheme as adjacent pairs of aqua molecules effectively bridge two sulfoxide residues. As seen from Fig. 2b), the 1,10-phenanthroline molecules project to either side of the supramolecular layer. The layers inter-digitate along [001], Fig. 2c), with the closest connections between layers being phenanthroline-C—H⋯O2(sulfate) interactions, Table 2Each of the aqua ligands donates two hydrogen bonds to different sulfate-O atoms, one of these hydrogen bonds is intramolecular while the remaining are intermolecular, Table 2Crystal Explorer 17 a). The overall contacts are also delineated into H⋯H, H⋯O/O⋯H, H⋯C/C⋯H and C⋯C contacts, as displayed in Fig. 6b)–(e), respectively. The short interatomic H⋯H contacts are characterized as the pair of beak-shaped tips at de + di ∼2.3 Å, Fig. 6b), and contribute 28.6% to the overall surface contacts. The significant O—H⋯O contacts between the aqua- and sulfate-O atoms make the major contribution to the overall contacts (44.5%), and these are represented as pairs of well-defined spikes at de + di ∼1.7 Å in Fig. 6c). The short interatomic H⋯C/C⋯H (19.5%) and C⋯C (5.7%) contacts are, respectively, characterized as pairs of broad symmetrical wings at de + di ∼2.9 Å in Fig. 6d), and the vase-shaped distribution of points at de + di ∼3.5 Å in Fig. 6e). The accumulated contribution of the remaining interatomic contacts is less than 2% and has a negligible effect on the packing.The overall two-dimensional fingerprint plot of (I)i.e. the electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) energy terms, after Turner et al.  are greater than for the C—H⋯O interaction involving the aqua-O atoms (−30.6 kJ mol−1). The energy frameworks were also computed and illustrate the above conclusions, Fig. 7In the present analysis, the pairwise interaction energies between the molecules in the crystal were calculated by summing up four different energy components,  al. 2017. These e al. 2017 while Ei.e. molecules conforming to the general formula fac-M(OH2)3OSO3. These include M = Mn (OH2)3OSO3 species, i.e. M = Mn  crystallizing in both forms. For the Mn complexes, the authors reporting the structure of the mer-isomer indicated that both forms were formed concomitantly from the slow evaporation of a methanol solution of the complex There are several literature analogues of (I)mer-isomer participates in three hydrogen bonds at the expense of the hydrogen bond involving the coordinated sulfate-O1 atom. The presence of inter-layer phenanthroline-C—H⋯O(sulfate) interactions persist as for the fac-isomer with the crucial difference that π–π stacking interactions are evident in the inter-layer region of the mer-form with the shortest separation being 3.76 Å.The key difference in the packing between the two isomers arises as one sulfate-O atom in the −3 being greater than 1.723 g cm−3 for the mer-isomer (OH2)3OSO3 complexes is the thermodynamically more stable form.The different packing arrangements result in different densities with that for (I)et al., 2019Given the isostructural relationship in the series (I)4·7H2O , 1,10-phenanthroline  and K(tcnoet)  in water–methanol ; where tcnoet is 1,1,3,3-tetracyano-2-ethoxypropenide. The mixture was sealed in a Teflon-lined autoclave and held at 403 K for 2 days, and then cooled to room temperature at a rate of 10 K h−1; yield: 35%. Light-pink blocks of the title complex suitable for single-crystal X-ray diffraction were selected directly from the synthesized product.The title compound was synthesized solvothermally under autogenous pressure from a mixture of CoSOUiso(H) set to 1.2Ueq(C). The oxygen-bound H atoms were located from a difference-Fourier map and refined with O—H = 0.84±0.01 Å, and with Uiso(H) set to 1.5Ueq(O). Owing to poor agreement, four reflections, i.e. (0 1 4), (0 0 2), (0 1 2) and (0 0 4), were omitted from the final cycles of refinement. The absolute structure was determined based on differences in Friedel pairs included in the data set.Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989020006271/hb7911sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020006271/hb7911Isup2.hklStructure factors: contains datablock(s) I. DOI: 2002737CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title unsymmetrical tertiary amine, which arose from the ring-opening reaction of a dihydrobenzoxazine, two 2,4-dimethylphenol moieties are linked by a 6,6′-(cyclohexylazanediyl)-bis(methylene) bridge: the dihedral angle between the phenol rings is 72.45 (7)°. The cyclohexyl ring adopts a chair conformation with the exocyclic C—N bond in an equatorial orientation. 24H33NO2, which arose from the ring-opening reaction of a dihydrobenzoxazine, two 2,4-dimethylphenol moieties are linked by a 6,6′-(cyclohexylazanediyl)-bis(methylene) bridge: the dihedral angle between the dimethylphenol rings is 72.45 (7)°. The cyclohexyl ring adopts a chair conformation with the exocyclic C—N bond in an equatorial orientation. One of the phenol OH groups forms an intramolecular O—H⋯N hydrogen bond, generating an S(6) ring, and a short intramolecular C—H⋯O contact is also present. In the crystal, O—H⋯O hydrogen bonds link the molecules into C(10) chains propagating along the [100] direction. The Hirshfeld surface analysis of the title compound confirms the presence of these intra- and intermolecular interactions. The corresponding fingerprint plots indicate that the most significant contacts in the crystal packing are H⋯H (76.4%), H⋯C/C⋯H (16.3%), and H⋯O/O⋯H (7.2%).In the title unsymmetrical tertiary amine, C H-benzo[e]-1,3-oxaxines  can undergo a ring-opening polymerization reaction to form polybenzoxazines. As a result of various promising physical and chemical properties, polybenzoxazines have been studied by a number of workers –] moiety provides such hydrogen bonding as to interconnect with other materials . Several isomers of dihydro-benzoxazines can be formed by varying the heteroatomic positions within the dihydro-oxazine ring. Among the different isomers of dihydro-benzoxazines, only 3,4-dihydro-2H-benzo[e]-1,3-oxaxines leads to the formation of small molecules instead of polybenzoxazines  adopts a distorted trigonal pyramidal shape because of the expansion of the angles around N1 atom .Fig. 1para-positions with respect to the OH groups deviate the most from the calculated mean planes with deviations of 0.043 (2) for C8 and −0.033 (2) Å for C17. The cyclohexyl group adopts a regular chair conformation as seen from the C—C—C bond angles, which are in the range 109.14 (17)° to 111.59 (17)°. The hydrogen atom bonded to C19 (H19) is in the axial position to allow the bulkier group (N1 tertiary-amine nitrogen atom) to be located at the equatorial position.The non-hydrogen atoms of the 2,4-dimethylphenol moieties, namely C1–C8/O1 and C11–C18/O2, are almost planar  and their mean planes subtend a dihedral angle of 72.45 (7)°. The C atoms in the methyl groups in the S(6) graph-set motif , H1 points toward N1 to set up an intramolecular O—H⋯N hydrogen bond with an f Table 1. This tyt Table 1 The charC(10) chains in the crystal, propagating in the [100] direction  hydrogen-bonded loops like those formed in 6,6′-(methylazanediyl)bis(methylene)bis  phenol group in (I)on Fig. 2. Unlike para-positions of the phenol rings  hydrogen-bond motif just mentioned, while the O1 and O2 atoms of (I)The structure overlay of the title compound (green compound) and its structural isomer with only ethyl groups at the Crystal Explorer 17.5 software  ring, C(10) chain, and C—H⋯O interaction, respectively.To better understand and visualize the interactions within the crystal of the title compound, a Hirshfeld surface (HS) analysis . The C⋯C contact only participates slightly in the crystal packing as its individual contribution is only 0.1%. The other contacts, i.e., N⋯N, H⋯N/N⋯H, C⋯N/N⋯C, C⋯O/O⋯C, show no effect on the crystal packing due to the contribution of 0.0%.Fig. 5et al., 2016et al., 2009et al., 2012ortho substituents have also been reported, e.g., tert-butyl substituents  linked by a bis(methylene)aza bridge in the Cambridge Structural Database 2 2 2  linked by bis(methylene)aza bridge, the number of match structures is now reduced to 38 hits as only the N-substituted grouping can change. Examples of different N-substituents of the 6,6′-(azanediyl)bis(methylene)bis derivatives are –CH2H-benzo[e]oxazine, was prepared by a one-pot Mannich reaction  three times, followed by 10 ml of deionized water for three more times until the solution became neutral. The product was dried by anhydrous sodium sulfate. Then, the dichloromethane solvent was removed by a rotary evaporator and consequently the dihydro-benzoxazine monomer, 3-cyclohexyl-6,8-dimethyl-3,4-dihydro-2H-benzo[e]oxazine, (II), was collected.Firstly, the corresponding dihydro-benzoxazine monomer, 3-cyclohexyl-6,8-dimethyl-3,4-dihydro-An equimolar amount of 2,4-dimethylphenol was then mixed with (II) and the mixture was heated at 333 K overnight. After the reaction was complete, the yellow viscous liquid turned into a yellow solid, which was washed using diethyl ether, giving rise to a white precipitate of the title compound, which was separated from the yellow solution by decantation and rinsing with diethyl ether. The white precipitate was recrystallized from propan-2-ol solution to yield colourless blocks of (I)−1): 3384 , 1484 , 1451 , 1245 , 1199 , 858 ; Raman (cm−1): 3023 , 2942 , 1447 ; 1H NMR : 1.06–1.14 , 1.19 , 1.44 , 1.64 , 1.81 , 1.94 , 2.21 , 2.72 , 3.73 , 6.70 , 6.85 , 8.04 ; 13C NMR : 16.03 (–CH3), 20.61 (–CH3), 25.99 (Ccy), 26.35 (Ccy), 27.66 (Ccy), 51.64 (–CH2–NR2), 57.65 (Ccy—NR2), 122.01 (Ca), 124.94 (Ca), 128.57 (Ca), 128.65 (Ca), 131.03 (Ca), 152.27 (C—OH) . Elemental analysis: analysis calculated for C24H33NO2 (%): C 78.47; H 8.99; N 3.82; found: C 78.49; H 8.97; N 3.85. The good agreement  between the measured PXRD pattern of (I)supporting information.M.p. 425 K; FTIR  = 1.2Ueq(carrier) or 1.5Ueq(methyl C) was applied in all cases. The absolute structure of (I)Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020009184/hb7926sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020009184/hb7926Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020009184/hb7926sup3.docxNon-covalent interaction plot and powder diffraction plot. DOI: Click here for additional data file.10.1107/S2056989020009184/hb7926Isup4.cmlSupporting information file. DOI: 2014264CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Phosphatidic acid (PA) is the simplest phospholipid and is involved in the regulation of various cellular events. Recently, we developed a new PA sensor, the N-terminal region of α-synuclein (α-Syn-N). However, whether α-Syn-N can sense physiologically produced, endogenous PA remains unclear. We first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all eleven lysine residues with glutamine residues. Using confocal microscopy, we next verified that α-Syn-N, but not α-Syn-N-KQ, detected PA in macrophagic phagosomes in which PA is known to be enriched, further indicating that α-Syn-N can be used as a reliable PA sensor in cells. Finally, because PA generated during neuronal differentiation is critical for neurite outgrowth, we investigated the subcellular distribution of PA using α-Syn-N. We found that α-Syn-N, but not α-Syn-N-KQ, accumulated at the peripheral regions (close to the plasma membrane) of neuronal growth cones. Experiments using a phospholipase D (PLD) inhibitor strongly suggested that PA in the peripheral regions of the growth cone was primarily produced by PLD. Our findings provide a reliable sensor of endogenous PA and novel insights into the distribution of PA during neuronal differentiation. Notablyin vitro . In addiin vitro . These rIn the present study, we first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all (eleven) lysine residues with glutamine residues. We next confirmed that α-Syn-N, but not α-Syn-N-KQ, recognized PA in macrophagic phagosomes where PA is known to be enriched ,17, furtPA produced by PLD and DGK is linked to neurite outgrowth . However22.1The mouse anti-6 × His-tag (D291-3) antibody was purchased from Medical and Biological Laboratories . A peroxidase-conjugated goat anti-mouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories . A mouse anti-FLAG M2 antibody was obtained from Sigma-Aldrich . The Alexa Fluor 594 goat anti-mouse IgG (A11005) and Alexa Fluor 594 phalloidin (A12381) were purchased from Thermo Fisher Scientific . 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0/18:1-PC) was purchased from Sigma-Aldrich. 1,2-dioleoyl-sn-glycero-3-phosphate (18:1/18:1-PA) was purchased from Avanti Polar Lipids . Cholesterol was obtained from Wako Pure Chemical Industries .2.2NdeI/XhoI sites, and the pAcGFP-C1 vector ) at the EcoRI/SalI sites. p3 × FLAG-Myr-DGKζ was constructed by inserting a PCR fragment encoding the full-length human DGKζ isoform 1 into the N-myristoylation sequence-fused p3 × FLAG-CMV-7.1 vector (Sigma-Aldrich) at the EcoRI/SalI sites. The N-myristoylation sequence was inserted into the translation initiation site of 3 × FLAG in the p3 × FLAG-CMV-7.1 [EcoRI/SalI sites.p6 × His-SUMO-α-Syn-N and pAcGFP-α-Syn-N were generated previously . To cons-CMV-7.1 . p3 × FL2.32+-NTA affinity chromatography) of 6 × His-SUMO-tagged proteins were performed as described previously [The expression and purification (Nieviously . For the2.4The liposome co-sedimentation assay was performed as described previously . The pur2.5COS-7 cells were maintained and transfected as described previously .2.62. The cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) as described by the manufacturer. Opsonized beads used for phagocytosis induction were prepared by incubating 3.0 μm polystyrene latex beads (Sigma-Aldrich) and 2 mg/ml normal rabbit IgG  at room temperature, and then incubating with goat anti-rabbit IgG-Alexa Fluor 594 . The prepared opsonized beads were added to the dish containing RAW264 cells cultured for 24 h after transfection; the cells were incubated for 10 min at 37 °C and fixed by 4% paraformaldehyde.RAW264 macrophage cells were maintained in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin  at 37 °C in an atmosphere containing 5% CO2.7Neuro-2a neuroblastoma cells were maintained under the same conditions as RAW264 cells. The cells were transfected using Polyfect reagent  as described by the manufacturer. To induce neuronal differentiation, Neuro-2a cells were cultured in serum-free DMEM for 48 h and then fixed by 4% paraformaldehyde.2.8The cells were processed as described previously  and were2.9P < 0.05 was considered significant.Data are represented as the means ± SD or ± SEM and were analyzed by Student's t-test, Kolmogorov–Smirnov test or Kruskal-Wallis followed by Dunn's multiple comparison test for multiple comparisons using GraphPad Prism 8  to determine any significant differences. 33.1http://www.compbio.dundee.ac.uk/jpred/)) was nearly the same as that of α-Syn-N (E. coli and then purified by Ni2+-affinity chromatography. The molecular mass of 6 × His-SUMO-α-Syn-N-KQ (~29 kDa) was greater than that of 6 × His-SUMO-α-Syn-N (~22 kDa) (We first generated a negative control of α-Syn-N (α-Syn-N-KQ) by replacing all eleven lysine residues, which are predicted to be critical for PA binding , with gl α-Syn-N A. Next, ~22 kDa) B. This d~22 kDa) B and C.Fin vitro liposome sedimentation assay, AcGFP-α-Syn-N co-localized with Myr-3 × FLAG-DGKζ at the plasma membrane (plasma membrane/cytosol intensity ratio: 1.88 ± 0.07 (mean ± SEM)) in COS-7 cells as previously reported [in vitro and in cells.In addition to the reported , whereas3.2We next examined whether α-Syn-N recognizes PA in macrophagic phagosomes, in which PA is well known to be enriched ,17. As s3.3We recently demonstrated that PA is intensely produced in Neuro-2a neuroblastoma cells during neuronal differentiation . Moreove3.4We next determined what enzyme(s) contributed to the PA production. Neuro-2a cells were treated with a PLD inhibitor ). Although AcGFP-α-Syn-N was strongly localized in the peripheral regions of growth cones in the absence of FIPI (plasma membrane/cytosol intensity ratio: 1.66 ± 0.06 (mean ± SEM)), the inhibitor (750 nM) almost completely inhibited the peripheral localization of AcGFP-α-Syn-N (plasma membrane/cytosol intensity ratio: 1.18 ± 0.04 (mean ± SEM)) , indicating that PLD primarily contributed to PA production in the region. Moreover, the results demonstrated that the peripheral localization of AcGFP-α-Syn-N occurs in a PA production-dependent manner, strongly suggesting that α-Syn-N recognized endogenously generated PA. Because FIPI nearly completely inhibited the peripheral localization of α-Syn-N, the contribution of DGK to PA generation in the region is likely small.4in vitro  as a negative control by changing all (eleven) lysine residues, which are predicted to be important for PA binding , to glutin vitro B and C ain vitro D and E. in vitro A, α-Syn-in vitro , in whicAxon growth is driven by the forward movement of a growth cone, which consists of a central domain rich in microtubules and a peripheral domain enriched in actin filaments . The disFIPI almost completely inhibited the localization of α-Syn-N at the peripheral regions in neurite growth cones , indicatEven in the presence of FIPI, some α-Syn-N in the peripheral regions of the growth cone remained (plasma membrane/cytosol intensity ratio: 1.15 (not 1.00)) . Thus, ol-lactate dehydrogenase A selectively interacted with 18:0/20:4- and 18:0/22:6-PA . Thus, a PA species sensor panel composed of many PA-binding proteins that selectively and characteristically recognize a variety of PA species is useful. Such a PA sensor panel could efficiently sense PA species produced in various cells in response to diverse cell stimuli. The panel may be able to distinguish PA produced by different PA-generating enzymes, for example, PLD-produced PA (mainly 16:0/18:1-PA species), DGKδ-produced PA (18:0/22:6-PA) and DGKζ-produced PA (primarily 16:0/16:0-PA species) in neurons.α-Syn substantially bound to 18:1/18:1-PA, 16:0/18:1-PA and 16:0/16:0-PA . PLD hydIn the present study, we provided a reliable PA sensor, α-Syn-N, which recognizes endogenous PA in cells. Moreover, we visualized PA in neurite growth cones for the first time using α-Syn-N and demonstrated that the PA was mainly generated by PLD. Our findings using the α-Syn-N sensor provide novel insights into PA distribution during neuronal differentiation. α-Syn-N is a useful sensor for endogenous PA, which plays important roles in a variety of physiological and pathological events.The authors declare no conflicts of interest associated with the contents of this article.


N,N′-tetramethylene dibromide monohydrate is presented, along with details of an improved synthesis and NMR spectroscopic analysis.The crystal structure of the viologen 4,4′-dimethyl-2,2′-dipyridyl- 16H20N22+·2Br−·H2O (1) is a member of the class of compounds called viologens. Viologens are quaternary salts of dipyridyls and are especially useful as redox indicators as a result of their large negative one-electron reduction potentials. Compound 1 consists of a dication composed of a pair of 4-methylpyridine rings mutually joined at the 2-position, with a dihedral angle between the pyridine rings of 62.35 (4)°. In addition, the rings are tethered via the pyridine nitrogen atoms by a tetramethylene bridge. Charge balance is provided by a pair of bromide anions, which are hydrogen bonded to a single water molecule [DO⋯Br = 3.3670 (15) and 3.3856 (15) Å]. The crystal structure of 1, details of an improved synthesis, and a full analysis of its NMR spectra are presented.The title compound, C Viologens are quaternary salts of dipyridyls, which have proven useful as redox indicators as a result of their large negative one-electron reduction potentials  does not produce HBr, but the reaction is slow; after five h, about 50% of the starting dipyridyl was recovered. The quaternization of tertiary amines is known as the Menschutkin reaction .Spotswood & Tanzer 1967 give gen1 is shown in Fig. 1via the pyridine nitrogen atoms by a tetramethylene bridge. There are no unusual bond lengths or angles. As a result of the two bridges between the pyridine rings, 1 occurs as two optical isomers, and therefore provides an example of atropisomerism  and 3.3856 (15) Å] (Table 1The molecular structure of ] Table 1.pyridyl⋯Br and 3.6581 (18) to 3.7700 (19) Å for type Cmethylene⋯Br. For comparison, the standard van der Waals radii of C, H, and Br  include water of crystallization. The tetramethylene bridge is present in CSD entries HIJGAI ° in 1]. In PICGAM, the dihedral angle is 72.64°, presumably as a result of the increased rigidity of the tether.The most similar structures to et al. , two of which (C2 and C4) are barely separated. Quantitative measurement using inverse-gated decoupling with a long recycle delay (60 s) shows that the carbon signals are of equal intensity. The 1-D 13C DEPT (Distortionless Enhancement by Polarization Transfer) and 2-D multiplicity-edited 1H–13C HSQC (Heteronuclear Single Quantum Coherence) establish a ratio of 3:2:1 for CH, CH2, and CH3, respectively. Further analysis of 2-D 1H COSY (Correlation Spectroscopy) and 2-D 1H-13C HMBC (Heteronuclear Multiple-Bond Correlation spectroscopy) spectra led to the NMR assignments summarized in Table 31H–13C correlations related to these two carbons. These details together with the 2-D 1H–15N HMBC, which reveals stronger H2/N9 and H4/N9 cross-peaks than H1/N9, clearly establish a symmetric three-ring molecular structure, as shown in Fig. 3There are eight resonance signals in the A while the geminal one at 4.03 ppm to H6B. The 1-D selective homonuclear decoupling 1H NMR spectra led to the extraction of J-coupling constants between these methylene protons (Table 33J coupling exists between H6B and H7B (11.3 Hz), followed by a sizable 3J coupling between H6A and H7A (6.1 Hz). As a result of the complexity of the spectra, the 3J(HA6HB7) and 3J(HB6HA7) could not be determined, but were estimated to be less than 2 Hz. Also, the 11.1 Hz coupling between H7A and H7B was tentatively assigned to the geminal coupling rather than the one across the C7–C7′ bond.The stereospecific assignment of the methylene protons was achieved by a systematic recording of 1-D selective NOESY (Nuclear Overhauser Effect Spectroscopy) and COSY spectra. A stronger NOE was observed between the proton at 4.73 ppm and H1, and thus this resonance was assigned to H6s Table 3. A largeAll NMR spectra were recorded on a Bruker Ascend 700 MHz spectrometer equipped with a TXO cryoprobe at 298 K. Spectra were indirectly referenced to the deuterium lock frequency, set to 4.7 ppm.ca 413 K) and nitrobenzene (b.p. 483 K). The mixture was refluxed for about 5 h, during which time a heavy precipitate formed. After cooling, the crude material was filtered and washed with acetone to yield 1.1 g of a tan-colored powder. Paper electrophoresis of this material at pH 7.5 showed (via UV) a small amount of starting material at p caR zero and product at pR −2.2 (pR is movement relative to picric acid). Crystallization from methanol–acetone gave 0.5–0.6 g (ca 50%) of reddish crystals, m.p. 528–530 K [lit. 528–533 K; Spotswood & Tanzer (1967max(water) 271 nm. IR(Nujol): 3456, 3414, 3372, 1632, 1582, 1566, 1514 1312, 1159, 1032, 853 cm−1.The starting materials were standard commercial samples of 95-98% purity. 4,4′-Dimethyl-2,2′-dipyridyl  and 1,4-dibromobutane  were added to a mixture of 5 mL each of xylene , 0.98 Å (RCH3), and 0.99 Å (R2CH2). Water hydrogen coordinates were refined, but subject to a restraint on the O—H distances (SHELXL command SADI). Uiso(H) parameters were set to values of either 1.2Ueq or 1.5Ueq (RCH3 only) of the attached atom.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020011147/ex2036sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020011147/ex2036Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020011147/ex2036sup3.tifFigure S1. DOI: Click here for additional data file.10.1107/S2056989020011147/ex2036sup4.tifFigure S2. DOI: Click here for additional data file.10.1107/S2056989020011147/ex2036Isup5.cmlSupporting information file. DOI: 2023167CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In both structures, the alkyloxy and methylsulfanyl groups are rotated out of the plane of the respective heterocyclic ring. 2Cl2. 1,3-Dimethoxy-2-(methylsulfanyl)imidazolium tetrafluoridoborate (1), C6H11N2O2S+·BF4−, displays a syn conformation of its two methoxy groups relative to each other whereas the two benzyloxy groups present in 1,3-dibenzyloxy-2-(methylsulfanyl)imidazolium tetrafluoridoborate (2), C18H19N2O2S+·BF4−, adopt an anti conformation. In the molecules of 1 and 2, the methylsulfanyl group is rotated out of the plane of the respective heterocyclic ring. In both crystal structures, intermolecular interactions are dominated by C—H⋯F—B contacts, leading to three-dimensional networks. The tetrafluoridoborate counter-ion of 2 is disordered over three orientations (occupancy ratio 0.42:0.34:0.24), which are related by rotation about one of the B—F bonds.Two salts were prepared by methylation of the respective imidazoline-2-thione at the sulfur atom, using Meerwein’s salt (trimethyloxonium tetrafluoridoborate) in CH Compounds belonging to this class can be converted into important derivatives with useful biological activity, 1 and 2 were prepared by methylation of 1,3-dimethoxyimidazoline-2-thione , is straightforward. The title compounds  al. 1994 for the 1, the two methoxy groups adopt a syn conformation relative to each other, and the methylsulfanyl group is anti to each of the methoxy groups ° (for the ring involving O1 and C4) and of 76.8 (1)° (for the ring involving O2 and C5). The methylsulfanyl group (S1–C6) is rotated out of the heterocyclic plane and forms a dihedral angle of 62.5 (1)° with the mean plane of the heterocycle defined by atoms N1, C1, N2, C2, and C3.In the organic cation of ps Fig. 1. In cont1, the methylsulfanyl group (S1–C18) of 2 is rotated out of the plane of the heterocycle and forms a dihedral angle of 78.6 (1)° with the mean plane defined by the imidazole ring atoms . The arrangement of the two benzyloxy moieties in the cation of 2 relative to each other is anti , which are related by a rotation about the B1—F1 bond  and 1.721 (3) Å for 1 and 2, respectively] determined in this study are in good agreement with the mean value (1.735 Å) calculated from 82 pertinent C—S distances compiled in the Cambridge Structural Database  as well as both methylene  and two aromatic hydrogen atoms  of the cation. In contrast to 1, the methylsulfanyl group (H18C) is also involved in hydrogen-bonding interactions.Both structures display multiple C—H⋯F—B contacts which cross-link the ion pairs and result in three-dimensional networks Tables 1 and 2 ▸.et al., 1994et al., 2016et al., 2010et al., 2017N-heterocyclic carbene–chalcogen cations.In addition to the classic 1,3-dimethylimidazolium-2-methylsulfanylimidazolium iodide : Trimethyloxonium tetrafluoridoborate  was added to a solution of 1,3-dimethoxyimidazoline-2-thione  in CH2Cl2 (20 ml). The mixture was stirred for 18 h at room temperature, then the solvent was evaporated. The residue was dissolved in EtOH (3 ml) and cooled at 277 K, forming colourless single crystals. The crystalline product was filtered, washed with Et2O (2 × 5 ml) and dried. Yield: 0.53 g, m.p. 363 K. 1H NMR : δ 2.72 , 4.25 , 8.43  ppm 13C NMR : δ 16.3, 68.9 (2C), 118.0 (2C), 135.7 ppm IR (neat): ν 3153 (m), 3134 (m), 1552 (m), 1445 (m), 1287 (w), 1043 (vs), 1018 (vs), 937 (s), 754 (s), 734 (s), 689 (m), 672 (m), 619 (m), 520 (s) cm−1.1,3-Di(benzyloxy)-2-methylsulfanylimidazolium tetrafluoridoborate (2): Trimethyloxonium tetrafluoridoborate  was added to a solution of 1,3-di(benzyloxy)imidazoline-2-thione  in CH2Cl2 (8 ml) in a Teflon test tube under argon. The mixture was stirred for 3 d at room temperature, then the solvent was evaporated. The residue was dissolved in MeOH (15 ml), precipitated with Et2O (15 ml), filtered, washed with Et2O and dried to yield a colourless powder. Single crystals were obtained by slow evaporation from an MeOH solution: Yield 0.42 g (62%), m.p. 407 K. 1H NMR : δ 2.54 , 5.45 , 7.49 , 8.36  ppm 13C NMR : δ 16.4, 83.1 (2C), 118.9 (2C), 128.9 (4C), 130.2 (2C), 130.4 (4C), 131.8 (2C), 136.8 ppm IR (neat): ν 3167 (w), 3137 (w), 1550 (w), 1492 (w), 1457 (w), 1384 (w), 1354 (w), 1214 (w), 1057 (vs), 1039 (vs), 907 (m), 873 (m), 844 (m), 773 (s), 739 (s), 699 (s), 671 (s), 575 (m), 499 (m) cm−1.Uiso parameters were set to 1.5 Ueq(C) of the parent carbon atom. H atoms bonded to secondary carbon atoms (C—H = 0.99 Å), and H atoms bonded to C atoms in aromatic rings (C—H = 0.95 Å) were positioned geometrically and refined with Uiso set to 1.2 Ueq(C) of the parent carbon atom.Crystal data, data collection and structure refinement details are summarized in Table 32 displays disorder of the tetrafluoridoborate ion involving three distinct components. Therefore, distance restraints were applied for all chemically equivalent B—F and F⋯F distances and restraints on displacement parameters of the F atoms affected by disorder were applied.The structure of 10.1107/S2056989020003643/wm5549sup1.cifCrystal structure: contains datablock(s) 1, 2, global. DOI: 10.1107/S2056989020003643/wm55491sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989020003643/wm55492sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989020003643/wm55491sup4.cmlSupporting information file. DOI: 1989707, 1989706CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title compound, the dihedral angle between the fused pyrazole and pyridine rings is 3.81 (9)°. The benzene ring forms dihedral angles of 35.08 (10) and 36.26 (9)° with the pyrazole and pyridine rings, respectively. In the crystal, weak C—H⋯O hydrogen bonds connect molecules along [100]. 18H18ClN3O2S, the dihedral angle between the fused pyrazole and pyridine rings is 3.81 (9)°. The benzene ring forms dihedral angles of 35.08 (10) and 36.26 (9)° with the pyrazole and pyridine rings, respectively. In the crystal, weak C—H⋯O hydrogen bonds connect molecules along [100].In the title compound, C Molecules built around the pyrazolopyridine core structure exhibit diverse medicinal properties that include anti-microbial, anti-viral, anti-fungal, anti-hypertensive, analgesic, anti-cancer, anti-inflammatory, anti-Alzheimer’s, anti-diabetic, anti-nociceptive, anti-tuberculosis, and anti-leishmanial activities  group attached to the pyrazolopyridine moiety exhibits an (−)anticlinal conformation [N3—C7—C6—C3 = −141.96 (19)°], as does the methylthio (SCH3) group attached to the pyrazolopyridine unit [C11—S1—C12—C13 = −128.93 (15) °] while the –COOC2H5 group attached to the pyrazolopyridine moiety has an (+)anti-periplanar conformation  and the methyl group attached to the pyrazole ring (NCH3) [C10—N2—C9—C8: −178.42 (19)°]. The fused pyrazole and pyridine rings are not exactly planar, subtending a dihedral angle of 3.81 (9)°. The dihedral angle between the planes of the benzene and pyrazole rings is 35.08 (10)° and that between the benzene and pyridine rings is 36.26 (9)°.The title compound has pyrazole Table 1.et al., 2016a]pyridine-6-carboxylate; Naik et al. 2019et al. 2016a]pyridin-3-yl(phenyl)methanone; Ravi et al. 2017a]pyridine; Wu et al. 2016et al. 19962CH3 substituent in the title compound are comparable with those reported for FIZLEI. Similarly, the geometrical parameters of the –C6H4Cl unit in the title compound are comparable with those for in DAWKAQ and NADPIU. The bond lengths of the pyrazolopyridine scaffold of the title compound are closer to those in NADPIU. The pyrazolopyridine moiety (N1–N3/C7–C9/C12–C14) of the title compound is approximately plan, as is also observed for FIZLEI, ALAFID, DAWKAQ, NADPIU and ZOJWAW. Apart from the CSD database, two other important databases, namely Drug Bank  and ethyl 2-(bis(methylthio)methylene)-3-oxobutanoate  in toluene (5 ml) under a blanket of dry N2, a catalytic amount of trifluoroacetic acid  was added. The resulting mixture was refluxed for 12 h, while monitoring progress by TLC . After completion of the reaction, the resulting mixture was subjected to purification by column chromatography to furnish 182 mg of the title compound in 75% yield as a colourless solid, m.p. 415.85 K, Rf = 0.3 (hexane:ethyl acetate 99:01). A sample suitable for single-crystal X-ray analysis was obtained by recrystallization from dry methanol.To a solution of 3-(4-chlorophenyl)-1-methyl-1Uiso(H) = 1.2 Ueq(C) or 1.5 Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020002479/lh5946sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020002479/lh5946Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020002479/lh5946Isup3.cmlSupporting information file. DOI: 1977404CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: