A walk around the A3-coupling

In recent years, the transition-metal catalyzed three-component coupling of an aldehyde, an alkyne and an amine, commonly called A(3)-coupling, has been established as a convenient and general approach towards propargylamines. Furthermore, the A(3)-coupling has found a broad application as a key step in the construction of various nitrogen-containing heterocycles, biologically active compounds and natural products. Several interesting modifications of the A(3)-coupling as well as different tandem reactions involving A(3)-coupling have been developed. This tutorial review aims to highlight the current achievements in the field of A(3)-couplings and related transformations.     

Il17A Autoantibody Induced by Recombinant Protein Ag85A-Il17A

IL17A is a widely studied cytokine which plays an important role in autoimmune diseases as well as asthma. In this study, murine IL17a gene fragment was inserted into pET28a-Ag85A and it was expressed as a fusion protein named Ag85A-IL17A. Recombinant protein Ag85A-IL17A was expressed as an open reading frame of 1,287 bp, encoding a polypeptide of 429 amino acid residues with a predicted molecular mass of 50 kDa. It was expressed in Escherichia coli. BL21 and purified by metal affinity chromatography using a nickel–nitrilotriacetic acid column. Bioinformatics revealed that Ag85A-IL17A contained B cell epitopes of IL17A. Purified proteins Ag85A-IL17A and Ag85A were injected with adjuvants into mice then antibody responses in sera were measured by enzyme-linked immunosorbent assay and western blot assay. Experiments showed that purified protein Ag85-IL17A could bind to a standard IL17A antibody and could induce IL17A autoantibody in mice.     

Specific GABA(A) agonists and partial agonists

The GABA(A) receptor system is implicated in a number of neurological and psychiatric diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the hetero-pentameric GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP, or isoguvacine, which we developed in the initial stages of the project. Using recombinant GABA(A) receptors, functional selectivity was demonstrated for a number of compounds, including THIP, showing highly subunit-dependent potency and maximal response. In light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogs of 4-PIOL, a low-efficacy partial GABA(A) agonist derived from THIP, have been performed. In this connection, a series of GABA(A) ligands has been developed that exhibit pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effects. Very little information is available on direct-acting GABA(A) receptor agonists in clinical studies. However, the results of clinical studies on the effect of the partial GABA(A) agonist THIP on human sleep patterns show that the functional consequences of a direct-acting agonist are different from those seen after the administration of GABA(A) receptor modulators, such as benzodiazepines and barbiturates.     

Synthesis and biological activities of 4-phenyl-5-pyridyl-1,3-thiazole derivatives as selective adenosine A3 antagonists

To investigate the potency of an adenosine A3 receptor (A3AR) antagonist as an anti-asthmatic drug, a novel series of 4-phenyl-5-pyridyl-1,3-thiazole derivatives was synthesized and evaluated in human adenosine A1, A2A and A3 receptor and rat adenosine A3 receptor binding assays. From investigation of the SAR study, compound 7af was identified as a highly potent human and rat A3AR antagonist. This compound inhibited IB-MECA-induced plasma protein extravasation in the skin of rats and showed good oral absorption. Also, compound 7af significantly inhibited antigen-induced hyper-responsiveness to acetylcholine in actively sensitized Brown Norway rats. These results show that 4-phenyl-5-pyridyl-1,3-thiazole derivatives are potential candidates to enable the evaluation of A3AR antagonists. Further evaluation of this class of compounds may afford a novel anti-inflammatory agent such as an anti-asthmatic drug.     

The effects of anion variation and ligand derivatization on silver coordination networks based upon weaker interactions

This article presents a series of silver(I) coordination networks based upon nonchelating bidentate thioether ligands. Frameworks using AgOTs as the silver(I) starting material form two-dimensional frameworks and are quite stable as shown by differential scanning calorimetry/thermogravimetric analysis (DSC/TGA) data. The networks are sufficiently robust as to maintain the same layered motif when the basic skeleton of the ligand is sequentially derivatized with -OEt, OBu, and OHex groups. Crystal structures of the AgOTs complexes of the underivatized and bis(hexoxy) derivatives, compounds 5 and 8, respectively, are presented as well as powder X-ray diffraction (PXRD) data of the other complexes. For 5, C20H20S3O3Ag, crystal data are as follows: monoclinic, space group P2(1)/n, a = 11.8117(5) A, b = 7.8813(5) A, c = 22.3316(10) A, beta = 102.245(5) degrees, V = 2031.6(2) A(3), Z = 4. For 8, C30H44S3O6Ag, crystal data are as follows: triclinic, space group Ponebar a = 8.445(4) A, b = 10.855(5) A, c = 19.308(9) A, alpha = 84.53(1) degrees, beta = 78.76(1) degrees, gamma = 68.43(1) degrees V = 1613.9(13) A(3), Z = 2. Changing the silver(I) starting material to AgPF6 results in a shift to a one-dimensional structure, 9, as shown by X-ray crystallography and in highly compromised stability. For 9, C14H16S2N2PF6Ag, crystal data are as follows: monoclinic, space group P2/n, a = 11.9658(11) A, b = 3.9056(4) A, c = 19.6400(18) A, beta = 92.87(1) degrees, V = 916.70(15) A(3), Z = 4.     

Antibody purification by affinity chromatography based on small molecule affinity ligands identified by SPR-based screening of chemical microarrays

Libraries of small molecules were searched for Fc-fragment selective binders to a recombinant human antibody ("MDJ8″, IgG(1)-subtype, κ-light chain) via SPR-based screening of chemical microarrays. Identified hit structures were immobilised on NHS-activated Sepharose for the determination of MDJ8 binding and selectivity versus typical proteineous impurities represented by the spend cell culture supernatant. Columns were packed and the most promising ligands further characterized in terms of binding constants, binding kinetics, as well as dynamic and equilibrium binding capacities. The performance of the best ligand, 2A10, was compared to standard Protein A chromatography. Using ligand 2A10 antibody capture from unprocessed cell culture supernatants was possible at similar recovery yield (>90%), purity (>80%), and eluting concentration (approximately 1 g/L) as with Protein A. Affinity constants (K(d)) of 2A10 were an order of magnitude higher than for the Protein A material, but still in the nM-range, while maximum binding capacities and binding kinetics were in the same order of magnitude. Ligand 2A10 was also able to capture a murine monoclonal antibody, again with similar efficiency as Protein A, as well as a number of humanised therapeutic antibodies. Antibody elution from the 2A10 column was possible using the Protein A standard protocol, i.e. 100mM glycine HCl pH 3.0, but also at near physiological pH, when some organic solvent or modifiers were present. Ligand 2A10 thus constitutes a cheaper, more robust alternative to Protein A as possible generic antibody binder. Moreover, the outlined approach to ligand selection could in principle by used to create suitable affinity ligands for other high value biotech products.     

Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type,Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer’s Disease Pathology

Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer’s disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer’s disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.     

Kinetics of copper incorporation into a biosynthetic purple Cu(A) azurin: characterization of red, blue, and a new intermediate species

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu(A) cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu(A) sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu(A) site of a biosynthetic Cu(A) model, Cu(A) azurin (Cu(A)Az) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu(A) reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu(A) center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu(A)Az allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ~410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu(A)Az variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu(A) centers.     

Stabilization of new forms of the intermetallic phases beta-RENiGe2 (RE = Dy, Ho, Er, Tm, Yb, Lu) in liquid indium

Flux conditions using liquid indium bypass the thermodynamically stable structure and yield new forms of the phases RENiGe2 (RE = Dy, Er, Yb, Lu). The compounds crystallize in the orthorhombic Immm space group and possess the YIrGe2 structure type. Lattice parameters for ErNiGe2, DyNiGe2, YbNiGe2, and LuNiGe2 are a = 4.114(1) A, b = 8.430(2) A, c = 15.741(5) A; a = 4.1784(9) A, b = 8.865(2) A, c = 15.745(3) A; a = 4.0935(6) A, b = 8.4277(13) A, c = 15.751(2) A, and a = 4.092(1) A, b = 8.418(3) A, c = 15.742(5) A, respectively. These phases represent a new structural arrangement (beta) of the compound type RENiGe2 as another set of compounds with identical stoichiometry are known to adopt the orthorhombic Cmcm CeNiSi2 type structure (alpha). In this paper we report the crystal and electronic band structure of four new members of the YIrGe2 structure type, as well as an investigation of the relative thermodynamic stabilities of the two forms.     

Enhancement of Lithium Storage Performance of Carbon Microflowers by Achieving a High Surface Area

High‐surface‐area, nitrogen‐doped carbon microflowers (A‐NCFs‐4) assembled from porous nanosheets are prepared in a three‐step process: soft‐templating self‐assembly, thermal decomposition, and KOH activation. The hydrazine hydrate used in our experiment serves not only as a structure‐directing agent, but also as a nitrogen source. The resultant A‐NCFs‐4 has a hierarchical porous structure and its specific surface area is as high as 2309 m² g^?1. When used as anode, it exhibits a reversible capacity as high as 807 mAh g^?1 at 300 mA g^?1 after 100 cycles, and an excellent rate capability of 200 mAh g^?1 at a high current density of 8 A g^?1. Compared with unactivated counterpart, A‐NCFs‐4 exhibits a significantly improved lithium storage capacity and rate capability; this can be attributed to its unique structural characteristics and high surface area. The hierarchical micro‐/mesopore structure, high surface area, and nitrogen doping of A‐NCFs‐4 could guarantee fast mass transport for lithium species, enhance the A‐NCFs‐4/electrolyte contact area, shorten the lithium‐ion diffusion length, and accommodate strain induced by volume changes during the electrochemical reaction. The results indicate that the as‐prepared A‐NCFs‐4 could be a promising candidate as a high‐performance anode for lithium‐ion batteries.     

Synthesis of 2,3- and 3,4-methanoamino acid equivalents with stereochemical diversity and their conversion into the tripeptide proteasome inhibitor belactosin a and its highly potent cis-cyclopropane stereoisomer

A series of chiral 2,3- and 3,4-methanoamino acid equivalents of stereochemical diversity were designed and synthesized from our chiral cyclopropane units, using a diastereoselective Grignard addition with ( R)- or ( S)- t-butanesulfinyl imines as the key step. These equivalents were converted into the proteasome inhibitor belactosin A and its cis-cyclopropane stereoisomer. The unnatural cis-isomer was shown to be more than twice as potent as belactosin A as a proteasome inhibitor.     

Exploring organosilane amines as potent inhibitors and structural probes of influenza a virus M2 proton channel

We describe the use of organosilanes as inhibitors and structural probes of a membrane protein, the M2 proton channel from influenza A virus. Organosilane amine inhibitors were found to be generally as potent as their carbon analogues in targeting WT A/M2 and more potent against the drug-resistant A/M2-V27A mutant. In addition, intermolecular NOESY spectra with dimethyl-substituted organosilane amine inhibitors clearly located the drug binding site at the N-terminal lumen of the A/M2 channel close to V27.     

A Strategy for Specific Fluorescence Imaging of Monoamine Oxidase A in Living Cells

Monoamine oxidase (MAO) has two isoforms, MAO‐A and MAO‐B, which show different functions, and thus selective fluorescence imaging is important for biological studies. Currently, however, specific detection of MAO‐A remains a great challenge. Herein, we report a new strategy for specific imaging of MAO‐A through the design of fluorogenic probes combining the characteristic structure of an inhibitor of the target enzyme along with propylamine as a recognition moiety. The high specificity of our representative probe is demonstrated by imaging MAO‐A in different live cells such as SH‐SY5Y (high levels of MAO‐A) and HepG2 (high levels of MAO‐B), and further validated by western blot analyses. The superior specificity of the probe may enable the accurate detection of MAO‐A in complex biosystems. Importantly, the use of the characteristic structure of an inhibitor, as demonstrated in this work, may serve as a general strategy to design specific recognition moieties for fluorogenic probes for enzymes.     

Scalable synthesis of cortistatin A and related structures

Full details are provided for an improved synthesis of cortistatin A and related structures as well as the underlying logic and evolution of strategy. The highly functionalized cortistatin A-ring embedded with a key heteroadamantane was synthesized by a simple and scalable five-step sequence. A chemoselective, tandem geminal dihalogenation of an unactivated methyl group, a reductive fragmentation/trapping/elimination of a bromocyclopropane, and a facile chemoselective etherification reaction afforded the cortistatin A core, dubbed "cortistatinone". A selective Δ(16)-alkene reduction with Raney Ni provided cortistatin A. With this scalable and practical route, copious quantities of cortistatinone, Δ(16)-cortistatin A (the equipotent direct precursor to cortistatin A), and its related analogues were prepared for further biological studies.     

Desymmetrization of (R,R)-hexa-1,5-diene-3,4-diol via monofunctionalization and rhodium-catalyzed allylic substitution

A sequence of selective monoprotection and Rh-catalyzed enantioconservative allylic substitution is established as a desymmetrization strategy for C(2)-symmetric hexa-1,5-diene-3,4-diol. A benzyl protecting group and ethyl carbonate as a leaving group emerged as the most useful combination with respect to reproducibility, stereoselectivity, and yield. A remarkable deviation from the normally observed regiospecificity of Rh-catalyzed allylic alkylations was observed for unprotected carbonates. In this case, a linear, rather than a branched alkylation product was obtained exclusively.     

Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain

Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discriminate between βCA2 and βCA1, this newly developed method was able to detect the addition of common bovine A1A2 milk to A2A2 milk, as low as 1%. Besides the detection of A2A2 milk purity, the developed assay can also be implemented as a rapid phenotyping method at dairy farms to replace the more invasive DNA-based screening. Additionally, the developed method was capable of detecting the addition of common bovine milk up to 1% in sheep, goat, buffalo, horse and donkey milk, which conforms to EU recommendations. In conclusion, a newly developed multiplex method capable of reliably detecting the dilution of A2A2 milk of multiple species, with common bovine milk up to 1%, is presented.     

Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host

Erythromycin A is a potent antibiotic long-recognized as a therapeutic option for bacterial infections. The soil-dwelling bacterium Saccharopolyspora erythraea natively produces erythromycin A from a 55 kb gene cluster composed of three large polyketide synthase genes (each ~10 kb) and 17 additional genes responsible for deoxysugar biosynthesis, macrolide tailoring, and resistance. In this study, the erythromycin A gene cluster was systematically transferred from S. erythraea to E. coli for reconstituted biosynthesis, with titers reaching 10 mg/l. Polyketide biosynthesis was then modified to allow the production of two erythromycin analogs. Success establishes E. coli as a viable option for the heterologous production of erythromycin A and more broadly as a platform for the directed production of erythromycin analogs.     

新型双孔蛋白质离子交换剂的制备及性能研究

本文以甲基丙烯酸缩水甘油酯为单体，三聚异氰尿酸三烯丙酯和二乙烯基苯为交联剂，甲苯和正庚烷为有机致孔剂，用原位聚合的方法合成新水性高分子载体。胺化反应后，得到含二乙胺基羟丙基的阴离子交换剂（树脂A）。研究了不同的合成配比对该树脂吸附牛血清白蛋白行为的影响，从而获得一个较佳的合成配方。在此基础上，采用固体颗粒与有机溶剂联合致孔模式，以硫酸钠颗粒为固体致孔剂，制备新型双孔离子交换剂（树脂B）。比较了两种树脂的孔结构及其对牛血清蛋白的吸附行为。结果表明，树脂A和树脂B均具有较高的吸附容量和较好的机械性能。由于树脂B内含有流动相可以对流通过的大孔结构，因此同时还具有相对较好的吸附动力学性能。     

Interactions of endoglucanases with amorphous cellulose films resolved by neutron reflectometry and quartz crystal microbalance with dissipation monitoring

A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase.     

REAu2In4 (RE = La, Ce, Pr, Nd): polyindides from liquid indium

The series of compounds REAu2In4 (RE = La, Ce, Pr, Nd) crystallize from excess In as rod-shaped single crystals. All members adopt the orthorhombic space group Pnma with a = 18.506(2) A, b = 4.6865(6) A, and c = 7.3414(9) A for LaAu2In4, a = 18.514(3) A, b = 4.6624(8) A, and c = 7.389(1) A for CeAu2In4, a = 18.420(4) A, b = 4.6202(9) A, and c = 7.376(2) A for the Pr analogue, and a = 18.406(2) A, b = 4.6114(5) A, and c = 7.4073(7) A for NdAu2In4. The REAu2In4 series can be regarded as polar intermetallic phases composed of a complex [Au2In4]3- polyanion network in which the rare-earth ions are embedded. The [Au2In4]3- network features In tetramer units, which defines the compounds as polyindides. Magnetic measurements found no magnetic ordering down to 2 K for any of the compounds. In addition, LaAu2In4 was found to be Pauli paramagnetic with a small susceptibility. Ab initio density functional methods were used to carry out electronic structure calculations to explore the bonding, the role of gold, and the contributions of different atoms to the density of states near the Fermi energy. We find that the density of states decreases slowly near Ef and reaches a minimum at about 0.5 eV above Ef.