Gold-Based Coronands as Hosts for M3+ Metal Ions: Ring Size Matters.

The controlled, self-assembled synthesis of multinuclear coordination compounds can be performed via different approaches. Frequently, steric, geometric and/or electronic factors located at the ligand systems predefine the way in which metal ions can assemble them to large aggregates. For the compounds in the present paper, also the Pearson's acidities and preferred coordination geometries of the metal ions were used as organization principles. The ligand under study, 2,6-dipicolinoylbis(N,N-diethylthiourea), H2L1ethyl, possesses 'soft' sulfur and 'hard' nitrogen and oxygen donors. One-pot reactions of this compound with [AuCl(tht)] (tht = tetrahydrothiophene) and M3+ salts (M = Sc, Y, La, Ln, Ga, In) give products with gold-based {Au3(L1ethyl)3}3+ or {Au2(L1ethyl)2}2+ coronands, which host central M3+ ions. The formation of such units is templated by the M3+ ions and the individual size of the coronand rings is dependent on the ionic radii of the central ions in a way that small ions such as Ga3+ form a [Ga⊂{Au2(L1ethyl)2}]+ assembly, while larger ions (starting from Sc3+/In3+) establish neutral [M⊂{Au3(L1ethyl)3}] units with nine-coordinate central ions.

Investigating the Sequence Determinants of the Curling of Amyloid Fibrils Using Ovalbumin as a Case Study.

Highly ordered, straight amyloid fibrils readily lend themselves to structure determination techniques and have therefore been extensively characterized. However, the less ordered curly fibrils remain relatively understudied, and the structural organization underlying their specific characteristics remains poorly understood. We found that the exemplary curly fibril-forming protein ovalbumin contains multiple aggregation prone regions (APRs) that form straight fibrils when isolated as peptides or when excised from the full-length protein through hydrolysis. In the context of the intact full-length protein, however, the regions separating the APRs facilitate curly fibril formation. In fact, a meta-analysis of previously reported curly fibril-forming proteins shows that their inter-APRs are significantly longer and more hydrophobic when compared to straight fibril-forming proteins, suggesting that they may cause strain in the amyloid state. Hence, inter-APRs driving curly fibril formation may not only apply to our model protein but rather constitute a more general mechanism.

Cobalt(III) Metallacryptates and Their Guest Cation-Exchange in Solution Monitored by 59Co NMR.

One-pot reactions of the catechol-scaffolding aroylbis(N,N-diethylthiourea) H2Lcat with mixtures of CoCl2 and MCl (M+ = Cs+, Rb+, K+, Tl+, or NH4+) or with a Co(NO3)2/TlNO3 mixture lead to the self-assembly of a series of cationic Co(III) metallacryptates of the general formula [M ⊂ {Co2(Lcat)3}]+ (M+ = Cs+, Rb+, K+, Tl+, or NH4+). Crystalline PF6- salts were obtained after workup with (n-Bu4N)(PF6), and the single-crystal structures of all five metallacryptates have been determined. Depending on the nature of the guest cations, the directional interactions between guest cations and the metallacryptand {Co2(Lcat)3} are either weak coordination contacts or hydrogen bonds. The bonding mode and the size of the guest ions slightly influence the molecular skeleton of the host molecule. These small structural variations also exist in solution and could be detected by means of 59Co NMR spectroscopy, which is shown to be a unique tool for an easy characterization of such compounds. 59Co NMR chemical shifts are extraordinarily sensitive to the guest cation in the metallacryptates, and time-arrayed 59Co NMR experiments show that cation-exchange processes in biphasic organic/aqueous systems can be studied in detail. This leads to insights into the relative rates of cation exchange, as well as the relative conditional distribution coefficients of such Co(III) metallacryptates between the aqueous and organic phases. Thus, the extent and the relative rate of the NH4+ ion exchange in [NH4 ⊂ {Co2(Lcat)3}](PF6) by Cs+ and K+ ions across the organic/aqueous phase boundary at room temperature have been studied by in situ 59Co NMR experiments. Preliminary 59Co NMR experiments show that the K+ ion in [K ⊂ {Co2(Lcat)3}](PF6) can be removed by its competitive complexation with the highly potassium-selective [2.2.2]cryptand, to give a transient 59Co NMR signal of the relatively unstable "empty" {Co2(Lcat)3} complex, which slowly decomposes in solution.

Trinuclear CoIILnIIICoII Complexes (Ln = La, Ce, Nd, Sm, Gd, Dy, Er, and Yb) with 2,6-Dipicolinoylbis(N,N-diethylthiourea): Synthesis, Structures, and Magnetism.

One-pot reactions of 2,6-dipicolinoylbis(N,N-diethylthiourea) (H2L) with Co(CH3COO)2·4H2O and LnCl3, where Ln = La, Ce, Nd, Sm, Gd, Dy, Er, and Yb, in warm methanol in the presence of Et3N, give stable trinuclear complexes of the composition [LnCo2(L)2(µ1,3-OOCCH3)2X] ("CoLnCo" complexes), where X- = κ2-CH3COO- or Cl-. X-ray structure determinations reveal symmetric trinuclear complexes containing two organic ligands (L2-), two terminal CoII ions, and one central LnIII ion. The organic ligands coordinate equatorially to the two CoII ions via two bidentate (O,S) N-acylthiourea moieties and tridentate to the central Ln ion via the (O,N,O) 2,6-dipicolinoyl moieties. Two acetate bridges established between each of the terminal Co and central Ln ions complete the square-pyramidal coordination spheres of CoII. All products possess an additional chlorido ligand axially coordinated to the lanthanide except the gadolinium(III) and lanthanum(III) complexes, where bidentate acetato ligands are coordinated. Fitting the χmT versus T data of the "CoLaCo" complex gives the axial and rhombic zero-field-splitting parameters D = 24.3(4) cm-1 and E = -1.0(2) cm-1, respectively, and anisotropic Landé values gx,y = 2.81(1) and gz = 2.00 as well as weak antiferromagnetic interactions between two high-spin CoII centers with J = -0.49(2) cm-1. The nature of the magnetic interactions between the LnIII ions and the CoII ions in the "CoLnCo" complexes is deduced by comparing their χMT values to the sum of χMT values of the analogous "CoLaCo" and related "ZnLnZn" complexes. The "CoDyCo" complex reveals an antiferromagnetic interaction, while the remaining "CoLnCo" complexes show ferromagnetic interactions.

In vivo biotinylated calpastatin improves the affinity purification of human m-calpain.

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies.

Iron(III) Metallacryptand and Metallacryptate Assemblies Derived from Aroylbis(N,N-diethylthioureas).

The reaction of isophthaloylbis(N,N-diethylthiourea), H2L1, with FeCl3·6H2O gives the dinuclear tris-complex [Fe2(L1)3] (5), possessing a cryptand-like structure. A similar reaction with the ligand 2,6-dipicolinoylbis(N,N-diethylthiourea), H2L2, however, results in the formation of the anionic, mononuclear Fe(III) complex [Fe(L2)2]- (6), which could be isolated as its "Tl+ salt" by the subsequent addition of Tl(NO3). A tighter view to the solid state structure of the obtained product, however, characterizes compound 6 as a one-dimensional coordination polymer, in which four-coordinate Tl+ ions connect the {[Fe(L2)2]-} units to infinite chains. When Fe3+ ions and Tl+ ions are added to H2L2 simultaneously in a one-pot reaction, a different product is obtained: a cationic trinuclear complex of the composition {M⊂[Fe2(L2)3]}+. It has been isolated as a PF6- salt and represents a {2}-metallacryptate with a nine-coordinate Tl+ ion in the central void. Structurally related products of the compositions {M⊂[Fe2(L2)3]}(PF6) (M = Na+, K+, Rb+) (8(PF6)) could be isolated from analogous reactions with alkaline salts instead of Tl(NO3). {2}-Metallacryptates with larger central voids were synthesized with the ether-spaced aroylbis(N,N-diethylthiourea) H2L3. The compounds {M⊂[Fe2(L3)3]}(PF6) (M = K+, Rb+, Tl+ or Cs+) (9(PF6)) were prepared by a similar protocol like those with H2L2 with the simultaneous addition of the metal ions to a solution of H2L3. Due to the larger spacer between the aroylthiourea units, the coordination number of the central M+ ions is 12 by six carbonyl and six ether oxygen atoms. All products were characterized by elemental analysis, IR spectroscopy, and X-ray structure analysis. Cyclic voltammetric studies were carried out with the three representative complexes [Fe2(L1)3], {K⊂[Fe2(L2)3]}(PF6), and {K⊂[Fe2(L3)3]}(PF6). The obtained voltammograms indicate the dependence of the redox properties of the oligonuclear systems on the conjugation in the organic backbones of the ligands.

Rhenium and Technetium Complexes with Pentadentate Thiocarbamoylbenzamidines: Steps toward Bioconjugation.

Thiocarbamoylbenzimidoyl chlorides, PhC(Cl)═N-(C═S)-NR(1)R(2), react with 2-(iminodiacetic acid)benzylamine under formation of the potentially pentadentate ligands H3L (R(1), R(2) = Et) and H3L-COOEt (R(1) = Me, R(2) = C6H4-4-COOEt) in high yields. Hydrolysis of H3L-COOEt in NaOH/MeOH gives quantitatively another benzamidine ligand H3L-COOH. The novel ligands readily react with (NBu4)[MOCl4] (M = Re, Tc) under formation of stable complexes with the general composition [MO(L)], in which they are triply deprotonated and fully occupy the remaining five coordination positions of the {MO}(3+) cores. In a "proof-of-principle" reaction for possible bioconjugations, the complex [ReO(L-COOH)] has been labeled with triglycine ethyl ester in high yields.

Potential for Aedes aegypti Larval Control and Environmental Friendliness of the Compounds Containing 2-Methyl-3,4-dihydroquinazolin-4-one Heterocycle.

2-Methylquinazolin-4(3H)-one was prepared by the reaction of anthranilic acid, acetic anhydride, and ammonium acetate. The reaction of 2-methylquinazolin-4(3H)-one with N-aryl-2-chloroacetamides in acetone in the presence of potassium carbonate gave nine N-aryl-2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide compounds. The structures of these compounds were elucidated on the basis of their IR, 1H nuclear magnetic resonance (NMR), 13C NMR, and high-resolution mass spectrometry (HR-MS) spectral data. These synthesized compounds containing the 2-methyl-3,4-dihydroquinazolin-4-one moiety exhibited activity against Aedes aegypti mosquito larvae with LC50 values of 2.085-4.201 µg/mL after 72 h exposure, which is also confirmed using a quantitative structure-activity relationship (QSAR) model. Interestingly, these compounds did not exhibit toxicity to the nontarget organism Diplonychus rusticus. In silico molecular docking revealed acetylcholine binding protein (AChBP) and acetylcholinesterase (AChE) to be potential molecular targets. These data indicated the larvicidal potential and environmental friendliness of these N-aryl-2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide derivatives.

Neutral gold complexes with tridentate SNS thiosemicarbazide ligands.

Na[AuCl(4)]·2H(2)O reacts with tridentate thiosemicarbazide ligands, H(2)L1, derived from N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chloride and thiosemicarbazides under formation of air-stable, green [AuCl(L1)] complexes. The organic ligands coordinate in a planar SNS coordination mode. Small amounts of gold(I) complexes of the composition [AuCl(L3)] are formed as side-products, where L3 is an S-bonded 5-diethylamino-3-phenyl-1-thiocarbamoyl-1,2,4-triazole. The formation of the triazole L3 can be explained by the oxidation of H(2)L1 to an intermediate thiatriazine L2 by Au(3+), followed by a desulfurization reaction with ring contraction. The chloro ligands in the [AuCl(L1)] complexes can readily be replaced by other monoanionic ligands such as SCN(-) or CN(-) giving [Au(SCN)(L1)] or [Au(CN)(L1)] complexes. The complexes described in this paper represent the first examples of fully characterized neutral Gold(III) thiosemicarbazone complexes. All the [AuCl(L1)] compounds present a remarkable cell growth inhibition against human MCF-7 breast cancer cells. However, systematic variation of the alkyl groups in the N(4)-position of the thiosemicarbazone building blocks as well as the replacement of the chloride by thiocyanate ligands do not considerably influence the biological activity. On the other hand, the reduction of Au(III) to Au(I) leads to a considerable decrease of the cytotoxicity.

Bipodal acylthiourea ligands as building blocks for Bi-, tetra-, and polynuclear oxorhenium(V) complexes.

Reactions of (NBu4)[ReOCl4] and 3,3,3',3'-tetraalkyl-1,1'-isophthaloylbis(thioureas), H2phth(R2tu)2 where R = Et, i-Bu, in hot MeOH with the addition of Et3N give red products of the composition [ReO(OMe){phth(R2tu)2}]2 (8a, R = Et; 8b, R = i-Bu). X-ray structures of 8 reveal symmetric binuclear complexes containing two almost coplanar organic ligands, each of which coordinates to two rhenium centers via the two bidentate-O,S moieties. The octahedral coordination spheres of the rhenium atoms are completed by each one oxo and one methoxido ligand which are directed perpendicular to the plane defined by the {phth(R2tu)2}(2-) ligands. While in 8a, both methoxido ligands point to the same side of the described plane and form a syn isomer, the MeO(-) ligands in 8b are located at opposite sides and form an anti isomer. Studies in solution show that there exists a reversible equilibrium between the anti and syn isomers. Dimerization/condensation of complexes 8 with the formation of tetranuclear complexes of the composition [{ReO{phth(R2tu)2}}2O]2 (9) and/or polynuclear species is observed in solutions, which do not contain MeOH.

A new labdane-type diterpenoid from the leaves of Vitex negundo L.

A new labdane-type diterpenoid, named vitexnegundin (1), along with seven known compounds, including vitexilactone (2), vitetrifolin D (3), 13-hydroxy-5(10),14-halimadien-6-one (4), (rel 3S,5S,8R,9R,10S)-3,9-dihydroxy-13(14)-labden-16,15-olide (5), artemetin (6), vitexcarpin (7) and penduletin (8), were isolated from the leaves of Vitex negundo L. Their structures were elucidated by using spectroscopic methods, X-ray crystallographic analysis and comparison with those reported in the literature. Moreover, all isolated compounds 1-8 were evaluated for their antimicrobial activity against ESBL-producing Escherichia coli strain and methicillin-resistant Staphylococcus aureus.

Targeting an Intrinsically Disordered Protein by Covalent Modification.

Intrinsically disordered proteins (IDPs) play important roles in the regulation of cellular function and in disease, and thus they represent an important group of therapeutic targets. Yet, members of this "disorderome" have not yet been successfully targeted by drugs, primarily because traditional design principles cannot be applied to their highly dynamic, heterogeneous structural states. Binders developed against IDPs so far suffer from very weak binding and inability to act in a cellular context. Here, we describe a possible generic method for the targeting of IDPs via covalent modification, which could entail specific and strong binding and inhibitory potential, making such "warheads" reasonable starting points of drug-development efforts. We demonstrate this principle by addressing the cysteine-specific covalent modification of calpastatin, the IDP inhibitor of the calcium-dependent cysteine protease calpain. We describe the protocol for monitoring the covalent modification of the inhibitor, measuring the Ki of its inhibition and comparing it to the Kd of its interaction with the enzyme. Our premise is that the underlying principles can be easily adapted to screen for molecules targeting other, disease-related, IDPs in the future.

Crystal structure of a tripeptide biphenyl hybrid C50H56N6O10·0.5H2O.

A peptide biphenyl hybrid compound {systematic name: dimethyl 2,2'-[((2S,2'S)-2,2'-{[(2S,2'S)-1,1'-([1,1'-biphen-yl]-2,2'-dicarbon-yl)bis-(pyrrolidine-1,2-diyl-2-carbon-yl)]bis-(aza-nedi-yl)}bis-(3-phenyl-propano-yl))bis-(aza-nedi-yl)](2S,2'S)-dipropionate hemihydrate}, C50H56N6O10·0.5H2O, was prepared by coupling of [1,1'-biphen-yl]-2,2'-dicarbonyl dichloride, tri-ethyl-amine and the tripeptide Pro-Phe-Ala in CH2Cl2 at 273 K under an N2 atmosphere. In the crystal, the asymmetric unit contains the peptide biphenyl hybrid accompanied by one-half of a water mol-ecule. A C atom of one of the proline rings is disordered between two positions in a 0.746 (11):0.254 (11) ratio. An important structural aspect of peptide compounds is their capacity to self-associate mediated by inter-molecular and intra-molecular hydrogen bonding. This characteristic can be useful in understanding the inter-actions between peptides and biomacromolecular targets, as well as to explain peptide properties.

Subnudatones A and B, new trans-decalin polyketides from the cultured lichen mycobionts of Pseudopyrenula subnudata.

Chemical investigation of the cultured polyspore-derived mycobionts of a Pseudopyrenula subnudata lichen led to the isolation of two new compounds, subnudatones A and B (1 and 2), together with four known compounds, 1-(2-hydroxy-1,2,6-trimethyl-1,2,4a,5,6,7,8,8a-octahydronaphthalen-1-yl)ethanone (3), libertalide C (4), aspermytin A (5), and 6,7-dimethoxy-4-hydroxymellin (6). Their chemical structures were elucidated by extensive 1D and 2D NMR analysis and high resolution mass spectroscopy, and comparisons were made with the literature. The absolute configuration of 1 was defined unambiguously using single crystal X-ray crystallography. Compound 1 represents the first dimeric decalin polyketide to be found in nature. The in vitro cytotoxicity of 1 against two cancer cell lines (K562 and MCF-7) was evaluated. Compound 1 showed moderate cytotoxic activity with IC50 values of 23.5 ± 1.0 and 51.9 ± 1.4 µM, respectively.

Oxotechnetium(V) complexes with a novel class of tridentate thiosemicarbazide ligands.

A novel tridentate thiosemicarbazide-type ligand class with an SNS donor set, H(2)L(1), was prepared by reactions of N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chlorides with thiosemicarbazides. H(2)L(1) ligands readily react with (NBu(4))[TcOCl(4)] in MeOH under the formation of red oxotechnetium(V) complexes of composition [TcOCl(L(1))]. The monomeric, five-coordinate compounds are air-stable and bind (L(1))(2-) tridentate in the equatorial coordination sphere. The compounds represent the first examples of oxotechnetium(V) complexes with NS chelate-bonded thiosemicarbazones.

Synthesis, structural characterization, and biological evaluation of oxorhenium(V) complexes with a novel type of thiosemicarbazones derived from N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chlorides.

Reactions of N-[N',N'-diethylamino(thiocarbonyl)]benzimidoyl chloride with 4,4-dialkylthiosemicarbazides give a novel class of thiosemicarbazides/thiosemicarbazones, H(2)L, which causes a remarkable reduction of cell growth in in vitro experiments. These strong antiproliferative effects are also observed for oxorhenium(V) complexes of the general composition [ReOCl(L)], which are formed by reactions of the potentially tridentate ligands with (NBu(4))[ReOCl(4)]. A systematic substitution of the alkyl groups in the thiosemicarbazone building blocks of the ligands do not significantly influence the biological activity of the metal complexes, while the replacement of the chloro ligand by a PPh(3) ligand (by the replacement of the oxo unit by a nitrido ligand) completely terminated the cytotoxicity of the metal complexes.

Calpain Purification Through Calpastatin and Calcium: Strategy and Procedures.

This chapter describes the strategy and procedures for the calcium-mediated affinity purification of calpain. The affinity capture method exploits the reversible binding properties of calpain's intrinsically disordered protein (IDP) inhibitor, calpastatin. IDPs are easily produced in heterologous expression hosts and purified to homogeneity. Combining these properties with in vivo biotinylation leads to a simplified purification strategy whereby biotinylated human calpastatin domain 1 (hCSD1) can capture calpain efficiently from a complex biological mixture with only a single chromatographic step and in a considerably reduced time. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest in order to capture its binding partner in a calcium- and chelator-based protocol.

Antimicrobial metabolites from a marine-derived Actinomycete Streptomyces sp. G278.

Analysis of an antimicrobial extract prepared from culture broth of the marine-derived actinomycete Streptomyces sp. G278 led to the isolation of ten compounds, 1-10. Two compounds, 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene (1), and 3-hydroxyl-2-methylpyridine (2) were isolated from a natural source for the first time. The structures of the isolated compounds were established by their spectral data analysis, including mass spectrometry, 1D-NMR, 2D-NMR, and X-ray crystallographic analysis in case of compound 3. All isolated compounds were evaluated for their antimicrobial activity against a panel of clinically significant microorganisms. Compounds 1 and 3 selectively inhibited Enterococcus faecalis (MIC: 256 µg/mL). Compound 2 was found to have antibacterial and antifungal activity against Escherichia coli (MIC: 64 µg/mL), Salmonella enterica (MIC: 256 µg/mL), Staphylococcus aureus (MIC: 256 µg/mL), Enterococcus faecalis (MIC: 256 µg/mL), and Candida albicans (MIC: 64 µg/mL). Except for compounds 9 and 10, the other known metabolites (4-8) also exhibited antimicrobial activity.

Two new triterpenoids from the roots of Phyllanthus emblica.

Two new triterpenes, the seco-friedelane type secofriedelanophyllemblicine and the ursane-derived saponin ursophyllemblicoside were isolated from the roots of the edible fruit-producing Phyllanthus emblica. Their structures were unambiguously elucidated using extensive 1D and 2D NMR analyses, high resolution mass spectrometry and single-crystal X-ray crystallographic analyses along with comparison with literature data. Secofriedelanophyllemblicine represents the first 3,4-secofriedelane bearing a carboxylic acid group substituent at C-20. Ursophyllemblicoside, incorporating the rare 21α hydroxyursolic acid as a sapogenol represents the first example of saponin comprising this aglycone. Secofriedelanophyllemblicine displayed a moderate cytotoxicity against K562 and HepG2 cancer cell lines.

Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin.

Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies.