Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development

Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western? (or Simon?) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon? can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.     

Determination of flavanol and procyanidin (by degree of polymerization 1-10) content of chocolate, cocoa liquors, powder(s), and cocoa flavanol extracts by normal phase high-performance liquid chromatography: collaborative study

An international collaborative study was conducted on an HPLC method with fluorescent detection (FLD) for the determination of flavanols and procyanidins in materials containing chocolate and cocoa. The sum of the oligomeric fractions with degree of polymerization 1-10 was the determined content value. Sample materials included dark and milk chocolates, cocoa powder, cocoa liquors, and cocoa extracts. The content ranged from approximately 2 to 500 mg/g (defatted basis). Thirteen laboratories representing commercial, industrial, and academic institutions in six countries participated in the study. Fourteen samples were sent as blind duplicates to the collaborators. Results from 12 laboratories yielded repeatability relative standard deviation (RSDr) values that were below 10% for all materials analyzed, ranging from 4.17 to 9.61%. The reproducibility relative standard deviation (RSD(R)) values ranged from 5.03 to 12.9% for samples containing 8.07 to 484.7 mg/g. In one sample containing a low content of flavanols and procyanidins (approximately 2 mg/g), the RSD(R) was 17.68%. Based on these results, the method is recommended for Official First Action for the determination of flavanols and procyanidins in chocolate, cocoa liquors, powder(s), and cocoa extracts.     

High toxicity and specificity of the saponin 3-GlcA-28-AraRhaxyl-medicagenate, from Medicago truncatula seeds, for Sitophilus oryzae

Background

Because of the increasingly concern of consumers and public policy about problems for environment and for public health due to chemical pesticides, the search for molecules more safe is currently of great importance. Particularly, plants are able to fight the pathogens as insects, bacteria or fungi; so that plants could represent a valuable source of new molecules.

Results

It was observed that Medicago truncatula seed flour displayed a strong toxic activity towards the adults of the rice weevil Sitophilus oryzae (Coleoptera), a major pest of stored cereals. The molecule responsible for toxicity was purified, by solvent extraction and HPLC, and identified as a saponin, namely 3-GlcA-28-AraRhaxyl-medicagenate. Saponins are detergents, and the CMC of this molecule was found to be 0.65?mg per mL. Neither the worm Caenorhabditis elegans nor the bacteria E. coli were found to be sensitive to this saponin, but growth of the yeast Saccharomyces cerevisiae was inhibited at concentrations higher than 100???g per mL. The purified molecule is toxic for the adults of the rice weevils at concentrations down to 100???g per g of food, but this does not apply to the others insects tested, including the coleopteran Tribolium castaneum and the Sf9 insect cultured cells.

Conclusions

This specificity for the weevil led us to investigate this saponin potential for pest control and to propose the hypothesis that this saponin has a specific mode of action, rather than acting via its non-specific detergent properties.     

Efficient Green‐Light‐Emitting Electrochemical Cells Based on Ionic Iridium Complexes with Sulfone‐Containing Cyclometalating Ligands

A new approach to obtain green‐emitting iridium(III) complexes is described. The synthetic approach consists of introducing a methylsulfone electron‐withdrawing substituent into a 4‐phenylpyrazole cyclometalating ligand in order to stabilize the highest‐occupied molecular orbital (HOMO). Six new complexes have been synthesized incorporating the conjugate base of 1‐(4‐(methylsulfonyl)phenyl)‐1 H‐pyrazole as the cyclometalating ligand. The complexes show green emission and very high photoluminescence quantum yields in both diluted and concentrated films. When used as the main active component in light‐emitting electrochemical cells (LECs), green electroluminance is observed. High efficiencies and luminances are obtained at low driving voltages. This approach for green emitters is an alternative to the widely used fluorine‐based substituents in the cyclometalating ligands and opens new design possibilities for the synthesis of green emitters for LECs.     

Facile Synthesis of Bis(crown ether)benzils: Prospective Building Blocks for Metal Ion Sensors

A new facile synthetic route to benzils containing fragments of 12-crown-4, 15-crown-5, and 18-crown-6 by oxidation of corresponding stilbenes was developed. The first representative of a new family of fluorescent sensors was obtained by reaction of bis(15-crown-5)benzil with o-phenylenediamine. The latter exhibits great fluorescence enhancement upon association with K⁺ and Rb⁺ compared to Na⁺ and Cs⁺.     

Convenient preparations of racemic and enantiopure methyl 6-oxopipecolate

Short, convenient syntheses of racemic and enantiopure methyl 6-oxopipecolate are described, starting from either pipecolic acid or (S)-lysine respectively. The sequence for the latter compound relies upon improved methodology for the oxidation of C-6 of lysine.     

Influence of Side‐Chain Composition on Polythiophene Properties and Supramolecular Assembly of Anionic Polythiophene Derivatives

A series of 10 polythiophene derivatives is reported, in which each polymer has a different percentage of carboxylic acid‐bearing repeat units. The properties of these polymers are explored under acidic conditions, where the carboxylic acid moieties remain neutral, and under basic conditions, where the carboxylic acid units become anionic carboxylates. The properties that are examined for both solutions and films include UV–vis absorption spectroscopy, photoluminescence spectroscopy, and red‐edge optical band gaps. All the properties studied are strongly dependent both on protonation state and percentage of carboxylic acid/carboxylate side chains along the polymer backbone. The anionic form of each polythiophene derivative was also used in layer‐by‐layer film deposition with a cationic phosphonium polyelectrolyte. The film growth process was studied by spectroscopic techniques to assess the influence of side‐chain composition on the film growth and optical properties. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019     

The synthesis and reactivity of the heptanuclear dianion [Os7(CO)20]2− including the synthesis and structural characterizations of the compounds [Os7(CO)20(AuPEt)3 2] and [Os8(CO)20(η6-C6H6]

Reduction of the heptaosmium cluster [Os₇(CO)₂₁] With [Et₄N][NH₄) gives the cluster dianion [Os₇(CO)₂₀]^2–,1, in high yield. The reaction of the dianion with [AuPR ₃Cl] (R=Et or Ph) in the presence of TlPF₆ forms [Os₇((CO)₂₀(AuPR ₃)₂] [R=Et (2a);R = Ph(2b)] in 80% yield, while the corresponding reaction with (Os(C₆H₆)(CH₃CN)₃]²⁺ gives [Os₈(CO)₂₀ ( ⁶-C₆H₆)] (3) in reasonable yield (ca. 30%). The dianion,1, and the clusters2 and3 have been fully characterized by bout spectroscopic and crystallographic methods. The crystal structure of the [Ph₄P]⁺ salt of1 shows that the metals in the anion adopt a capped octahedral geometry, with all twenty carbonyl ligands in terminal sites. The metal core geometry in2a is best described as a tricapped octahedron, and is based on the structure of the dianion1 with two adjacent octahedral faces capped by the Au atoms of the two AuPEt₃ groups. In a similar fashion, the geometry of3 is related to that of1 with the addition of an Os(C₆H₆) unit capped to a triangular face, to give a bicapped octahedral framework.     

Dications of benzylidenefluorene and diphenylmethylidene fluorene: the relationship between magnetic and energetic measures of antiaromaticity

Oxidation of m- and p-substituted benzylidene fluorenes to antiaromatic dications was attempted by electrochemical and chemical means. Electrochemical oxidation to dications was successful for benzylidene fluorenes with p-methoxy, p-methyl, p-fluoro, and unsubstituted phenyl rings in the 3-position; attempts to oxidize the m-substituted derivatives via electrochemistry were unsuccessful. Chemical oxidation with SbF(5)/SO(2)ClF gave the dication of 9-[(4-methoxyphenyl)methylene]-9H-fluorene cleanly; oxidation of all other substituted benzylidene fluorenes resulted in mixtures of products. The excellent linear relationship between the chemical shifts calculated by the GIAO method and the experimental shifts for the p-methoxy-substituted benzylidene fluorene dication suggests that the calculations satisfactorily reflect the magnetic properties of this dication and potentially those of the other dications studied. The redox potentials from electrochemical oxidation, a measure of the stability of the dications, showed a good linear relationship with another measure of stability, the calculated difference in energy between each dication and its neutral precursor. The dications of benzylidene fluorenes were less stable than the dications of diphenylmethylidene fluorenes; within each type of compound, dications with p-substituted phenyl rings were more stable than dications with m-substituted phenyl rings and dications with phenyl rings substituted with electron-donating groups were more stable than dications with phenyl rings substituted with electron-withdrawing groups. The antiaromaticity of the fluorenyl system was assessed through the nucleus-independent chemical shift (NICS) that was also calculated by the GIAO method. The plot of the NICS values per square area versus the calculated energy difference for the dications showed a moderate degree of linearity; the plot of NICS values per square area versus the oxidation potentials was less linear. Thus, a suggestive, but not conclusive, relationship between magnetic and energetic measures of antiaromaticity was observed.     

Bidirectional "ping-pong" energy transfer and 3000-fold lifetime enhancement in a Re(I) charge transfer complex

The synthesis and photophysics of a new Re(I)-carbonyl diimine complex, Re(PNI-phen)(CO)(3)Cl, where the PNI-phen is N-(1,10-phenanthroline)-4-(1-piperidinyl)naphthalene-1,8-dicarboximide is reported. The metal-to-ligand charge transfer (MLCT) emission lifetime was increased approximately 3000-fold at room temperature with respect to that of the model complex [Re(phen)(CO)(3)Cl] as a result of thermal equilibrium between the emissive (3)MLCT state and a long-lived triplet ligand-centered ((3)LC) state on the PNI chromophore. This represents the longest excited state lifetime (τ = 651 μs) that has ever been observed for a Re(I)-based CT photoluminescence at room temperature. The energy transfer processes and the associated rate constants leading to the establishment of the excited state equilibrium were elucidated by a powerful combination of three techniques (transient visible and infrared (IR) absorption and photoluminescence), each applied from ultrafast to the micro/milliseconds time scale. The MLCT excited state was monitored by transient IR using CO vibrations through time intervals where the corresponding signals obtained in conventional visible transient absorption were completely obscured by overlap with strong transients originating from the pendant PNI chromophore. Following initial excitation of the (1)LC state on the PNI chromophore, energy is transferred to form the MLCT state with a time constant of 45 ps, a value confirmed in all three measurement domains within experimental error. Although transient spectroscopy confirms the production of the (3)MLCT state on ultrafast time scales, Fo?rster resonance energy transfer calculations using the spectral properties of the two chromophores support initial singlet transfer from (1)PNI* to produce the (1)MLCT state by the agreement with the experimentally observed energy transfer time constant and efficiency. Intersystem crossing from the (1)MLCT to the (3)MLCT excited state is believed to be extremely fast and was not resolved with the current experiments. Finally, triplet energy was transferred from the (3)MLCT to the PNI-centered (3)LC state in less than 15 ns, ultimately achieving equilibrium between the two excited states. Subsequent relaxation to the ground state occurred via emission resulting from thermal population of the (3)MLCT state with a resultant lifetime of 651 μs. The title chromophore represents an interesting example of "ping-pong" energy transfer wherein photon excitation first migrates away from the initially prepared (1)PNI* excited state and then ultimately returns to this moiety as a long-lived excited triplet which disposes of its energy by equilibrating with the photoluminescent Re(I) MLCT excited state.