Evaluation of cell-free protein synthesis using PDMS-based microreactor arrays.

A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.     

Fabrication optimization of a miniaturized array device for cell-free protein synthesis

Cell-free protein synthesis (CFPS) is an attractive alternative to cell-based protein expression systems because of its advantages including speed, simplicity, and adaptability to various formats. However, two major obstacles exist that have been preventing it from being widely used. One is high cost and the other is low protein synthesis yield. We report here a miniaturized CFPS device that addresses these challenges. The cost saving was achieved by miniaturization, which reduced the reagent consumption by two orders of magnitude. The protein synthesis yield was enhanced by prolonging CFPS reactions through continuous supply of reactants (e.g. nutrients and energy components). The reactants were contained in a feeding solution, which was replenished through a nanoporous membrane and microchannel. The design of the miniaturized device was optimized by running continuous-exchange CFPS in devices with a variation in the type of membrane, the size of the exchange interface, and the volume ratio of the reaction solution to the feeding solution. The effects of these design variations on the protein synthesis yield have been studied. Furthermore, the design was expanded into a 96-unit device that can produce a large number of proteins simultaneously, enabling high-throughput proteomics applications.     

Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS

We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.     

Microreactor with integrated temperature control for the synthesis of CdSe nanocrystals

The recent needs in the nanosciences field have promoted the interest towards the development of miniaturized and highly integrated devices able to improve and automate the current processes associated with efficient nanomaterials production. Herein, a green tape based microfluidic system to perform high temperature controlled synthetic reactions of nanocrystals is presented. The device, which integrates both the microfluidics and a thermally controlled platform, was applied to the automated and continuous synthesis of CdSe quantum dots. Since temperature can be accurately regulated as required, size-controlled and reproducible quantum dots could be obtained by regulating this parameter and the molar ratio of precursors. The obtained nanocrystals were characterized by UV-vis and fluorescence spectrophotometry. The band width of the emission peaks obtained indicates a narrow size distribution of the nanocrystals, which confirms the uniform temperature profile applied for each synthetic process, being the optimum temperature at 270 °C (full width at half maximum = 40 nm). This approach allows a temperature controlled, easy, low cost and automated method to produce quantum dots in organic media, enhancing its application from laboratory-scale to pilot-line scale processes.     

Ricinus communis toxin-mediated inhibition of protein synthesis in cell-free extracts of a toxin-resistant variant mouse lymphoma cell line.

Ricinus communis agglutinin II (RCAII, ricin, toxin) at low concentrations inhibits protein synthesis in cell-free extracts, but not in intact cells, of an RCAII-resistant mouse lymphoma variant cell line. The concentration dependence of the inhibition by RCAII was the same in cell-free extracts of both RCAII-resistant variant and RCAII-sensitive parental cells, while intact parental cells are 250 times more sensitive to RCAII toxicity. The onset of RCAII inhibition of cell-free protein synthesis was extremely rapid in both cases, being complete in a few minutes. Under these conditions RCAII inhibits protein synthesis in intact RCAII-sensitive parental cells, but maximal inhibition requires several hours to occur. These results support our previous electron microscopic observations that the variant cells are defective in the uptake of RCAII by endocytosis at low toxin concentrations.     

Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein–ligand complex structure analysis

Room-temperature (RT) protein crystallography provides significant information to elucidate protein function under physiological conditions. In particular, contrary to typical binding assays, X-ray crystal structure analysis of a protein–ligand complex can determine the three-dimensional (3D) configuration of its binding site. This allows the development of effective drugs by structure-based and fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein–ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein–ligand complex analysis, the simultaneous preparation of protein–ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein–ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein–ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin–ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.

Room temperature protein crystallography and its application to protein–ligand complex structure analysis was demonstrated using a microfluidic protein crystal array device.     

Electrokinetic characterization of hybrid NOA 81-glass microchips: Application to protein microchip electrophoresis with indirect fluorescence detection

A low-cost and straightforward hybrid NOA (Norland optical adhesive) 81-glass microchip electrophoresis device was designed and developed for protein separation using indirect fluorescence detection. This new microchip was first characterized in terms of surface charge density via electroosmotic mobility measurement and stability over time. A systematic determination of the electroosmotic mobility (μ_eo) over a wide pH range (2–10) and at various ionic strengths (20–50 mM) was developed for the first time via the neutral marker approach in an original simple frontal methodology. The evolution of μ_eo was proved consistent with the silanol and thiol functions arising from the glass and the NOA materials, respectively. The repeatability and reproducibility of the measurements on different microchips (RSD < 14%) and within 15 days (less than 5% decrease) were successfully demonstrated. The microchip was then applied for the efficient electrophoretic separation of proteins in a zonal mode coupled with indirect fluorescence detection, which is, to our knowledge, the first proof of concept of capillary zone electrophoresis in this hybrid microsystem.     

The oxazole-triamide rearrangement. Application to peptide synthesis

The carboxyl group of a N-acylated amino acid may be protected by conversion to an oxazole derivative which, on photoxygenation, regenerates the carboxyl group in activated (triamide) form for peptide synthesis.     

Application of commercial microwave ovens to organic synthesis.

Commercial microwave ovens have been safely used to dramatically reduce the reaction times (at comparable yield) of Diels-Alder, Claisen, and ene reactions. Significant solvent effects were also observed.     

A novel microfluidic flow-injection analysis device with fluorescence detection for cation sensing. Application to potassium

A microfabricated device has been developed for fluorimetric detection of potassium ions without previous separation. It is based on use of a fluorescent molecular sensor, calix–bodipy, specially designed to be sensitive to and selective for the target ion. The device is essentially made of a Y-shape microchannel moulded in PDMS fixed on a glass substrate. A passive mixer is used for mixing the reactant and the analyte. The optical detection arrangement uses two optical fibres, one for excitation by a light-emitting diode, the other for collection of the fluorescence. This system enabled the flow-injection analysis of the concentration of potassium ions in aqueous solutions with a detection limit of 0.5 mmol L⁻¹ and without interference with sodium ions. A calibration plot was constructed using potassium standard solutions in the range 0–16 mmol L⁻¹, and was used for the determination of the potassium content of a pharmaceutical pill. Figure Photography of the microfluidic channel showing the ridges in the PDMS substrate at the top of the channel     

Serendipitous synthesis of a cyclic formamidine. Application to the synthesis of polyazamacrocycles

An unexpected synthesis of 4,5,6,7,8,9-hexahydro-1,3-diazonine, using hexamethylenediamine and dimethylformamide, is presented. The cyclization process was explained as a two-step process involving formylation and subsequent intramolecular condensation. The scope of this process was demonstrated through the synthesis of two other nitrogen-containing macroheterocycles.     

Parham-type cycliacylation with Weinreb amides. Application to the synthesis of fused indolizinone systems

[reaction: see text] Weinreb amides behave as efficient internal electrophiles in Parham-type cycliacylation reactions. Thus, aryl- and heteroaryllithiums generated by lithium-halogen exchange undergo intramolecular cyclization to give fused indolizinone systems as pyrrolo[1,2-b]isoquinolines, thieno[2,3-f]indolizinones, and pyrrolo[1,2-b]acridinones in high yields.     

Characterization of modified lead titanate piezoceramics. Application to the design of array transducers

Due to their high electromechanical anisotropy, lead titanate piezoelectric ceramics of modified composition are very interesting for designing either linear or annular ultrasonic transducer arrays used in medical diagnosis and in non-destructive testing. In this paper results of the characterization of three varieties of this type of material are presented. The characterization includes the measurement of the resonance frequency dispersion diagram of thin parallelepipedic elements and the measurement of the characteristics in the longitudinal thickness mode and the planar mode. In view of the application to the design of transducers, several types of structures are presented.     

Tandem enantioselective conjugate addition--cyclopropanation. Application to natural products synthesis

A tandem asymmetric conjugate addition-cyclopropanation was developed, in which a cyclic or linear enone was converted to a TMS-protected 3-substituted-cyclopropanol in an efficient one-pot reaction. These compounds were then selectively cleaved to yield alpha-methyl-beta-alkyl ketones, alpha-methylene-enones, or chain extended gamma-alkyl-enones. This methodology was applied to the formal total synthesis of (-)-(S,S)-clavukerin A and (+)-(R,S)-isoclavukerin.     

Iron-catalyzed benzamide formation. Application to the synthesis of moclobemide

A convenient and user-friendly method to yield benzamides from primary and secondary amines and various benzylic alcohols in the presence of a cheap iron salt (FeCl₂·4H₂O) and tert-butylhydroperoxide (70% in water) as a stoichiometric oxidant is described. Control experiments indicated that this reaction might involve radical species. This method proved to be general, generating a family of 30 benzamides and was applied to the preparative synthesis of anti-anxiety drug moclobemide.     

2,3-Anhydrosugars in glycoside bond synthesis. Application to alpha-D-galactofuranosides

We report here the use of 2,3-anhydro-D-gulofuranosyl thioglycosides and glycosyl sulfoxides in the synthesis of alpha-D-galactofuranosidic bonds, which are present in a range of bacterial and fungal glycoconjugates. This two-step method involves a stereoselective glycosylation in which a 2,3-anhydro-alpha-D-gulofuranoside is obtained either as the sole or as the major product, followed by a regioselective opening of the epoxide ring using lithium benzylate in the presence of (-)-sparteine. In exploring the scope of the method, donors protected at O5 and O6 with an isopropylidene acetal, benzyl ethers, or benzoate esters were studied. Overall, the glycosyl sulfoxides provided the products in slightly higher yields and selectivity, with the best results being obtained with benzylated and benzoylated substrates. In the epoxide ring-opening reactions, the acetal- and ether-protected donors afforded poor to modest regioselectivity, whereas the benzoylated products gave good yields of the desired alpha-D-galactofuranosides. The benzoyl-protected species are, therefore, the donors of choice for these reactions. The utility of the approach was demonstrated through the synthesis of three alpha-D-galactofuranosyl-containing oligosaccharides.     

Convenient access to bis-indole alkaloids. Application to the synthesis of topsentins

Topsentins and related bis-indole alkaloids may be efficiently synthesized through an addition/oxidation sequence leading to 2-(3-indolylcarbonyl)-imidazole derivatives followed by a Pd-catalyzed heteroarylation with the appropriate 3-stannylindoles.