Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption

The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6 ± 3.1% of RNA, 94.6 ± 0.8% of proteins and 96.7 ± 11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2 ± 4.0%), even though the RNA (65.5 ± 2.8%), protein (84.4 ± 1.3%) and gDNA clearance (44.7 ± 14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.     

Determination of trace ethylene glycol in industrial solvents and lubricants using phenyl boronic acid derivatization and multidimensional gas chromatography

A practical gas chromatographic approach is introduced for the characterization of trace ethylene glycol in industrial solvents and lubricants. The analytical approach employs single step derivatization technique that effectively converts ethylene glycol to the cyclic boronate ester (2-phenyl-1,3,2-dioxaborolane), using phenyl boronic acid as a derivatizing reagent. The separation of the derivatized product was achieved by using multidimensional gas chromatography. Heavy lubricant matrices like engine crankcase oil were back-flushed to improve sample throughput and system cleanliness. Detection and quantitation of 2-phenyl-1,3,2-dioxaborolane was conducted with mass spectrometry in selected ion monitoring mode.     

基于叠氮基-氰基点击化学的苯硼酸亲和硅胶的制备及其在糖蛋白/糖肽选择性富集中的应用

硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性，生物相容性，制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题，设计并开发了一种新型苯硼酸亲和硅胶（TCNBA）。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成，生物相容性好，制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行评价，结果表明，TCNBA能够分别从辣根过氧化物酶（HRP）和免疫球蛋白G（IgG）酶解液中鉴定出13个和11个糖肽；以HRP和牛血清白蛋白（BSA）酶解液混合物（物质的量比1：10）作为研究对象，富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）进行评价，以HRP、IgG、核糖核酸酶B（RNaseB）作为考察对象，结果表明，TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示，富集后非糖蛋白得到较大程度去除，糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。     

Synthesis and analysis of thermal characteristics of polybenzoxazine based on phenol and 3‐Amino phenyl boronic acid

In this work, 3‐amino phenyl boronic acid (AB) was used as an aniline derivative in the preparation of polybenzoxazine based on phenol. In order to investigate the effect of boronic acid on thermal characteristics, polybenzoxazines based on pure aniline and 50% aniline and AB mixture were also prepared and analyzed. Significant improvements in thermal characteristics, increase in thermal stability and char yield, was recorded for the polymers based on AB or its mixture. This behavior was associated with crosslinked structures generated by condensation reactions of B? OH groups. Morphologic and thermal characteristics of polybenzoxazines samples were investigated by NMR, FTIR, DSC, TGA, and direct pyrolysis mass spectrometry (DP‐MS) techniques. Application of DP‐MS technique also supplied additional information on crosslinked structures produced by boronic acid units. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 1711–1716     

One saccharide sensor based on the complex of the boronic acid and the monosaccharide using electrochemical impedance spectroscopy

A novel saccharide sensor based on the covalent interaction between the boronic acid and saccharides was developed. Poly (aminophenylboronic acid) (PABA) was prepared by electropolymerizing 3-aminophenylboronic acid on gold electrode surface in acidic solution. The boronic acid group of the PABA film can form covalent-bond with different saccharides, which can change the dielectric characteristics of the PABA film, and the change of the dielectric characteristic was saccharides concentration dependent. Four kinds of saccharides could be detected by using electrochemical impedance spectroscopy. Good linear relationship and high sensitivity were obtained by this method.     

Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support

DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids <10 kbp, in the future larger plasmids would be required. The purpose of this work was to study the chromatographic behavior of nongrafted carbonyldiimidazole monolithic disks using plasmids with different sizes under hydrophobic conditions. Thereunto, the purification of several plasmids was performed. Higher size plasmids needed lower ammonium sulfate concentration, due to the greater number of interactions between the plasmids and monolith. The dynamic binding capacity experiments for the different plasmids revealed a lower capacity for bigger plasmids. It was also verified that the increase of salt concentration from 2.5 to 3 M of ammonium sulfate increased the capacity. At the highest salt concentration, a slight improvement in the capacity using lower flow rate was observed, possibly due to compaction of plasmid molecules and its better organization on the monolith channels. Finally, a low pH also had a positive effect on the capacity. So, this monolithic support proved to be appropriate to purify the supercoiled isoform of different plasmids with different sizes, providing a valuable instrument as a purification technique.     

Fluorescence sensors for monosaccharides based on the 6-methylquinolinium nucleus and boronic acid moiety: potential application to ophthalmic diagnostics

Continuous monitoring of glucose levels in human physiology is important for the long-term management of diabetes. New signaling methods/probes may provide an improved technology to monitor glucose and other physiologically important analytes. The glucose sensing probes, BMQBAs, fabricated using the 6-methylquinolinium moiety as a fluorescent indicator, and boronic acid as a chelating group, may have versatile applications in glucose sensing because of their unique properties. In this paper we discuss the design logic, synthesis, characterization and spectral properties of three new isomeric glucose sensors (BMQBAs), and a control compound (BMQ) in the presence and absence of sugars. The sensing ability of the new probes is based on a charge neutralization and stabilization mechanism upon sugar binding. The new probes have attractive fluorescence quantum yields, are highly water-soluble, and have spectral characteristics compatible with cheap and portable LEDs and LDs. One of the probes, o-BMQBA, has a sugar bound pK_a of 6.1, and a dissociation constant K_D of 100 mM glucose. These probes have been designed specifically to respond to tear glucose in a contact lens polymer for ophthalmic glucose monitoring, where the reduced sugar bound pK_a affords for sensing, in a lens environment that we have previously shown to be mildly acidic.     

Potential of boronic acid functionalized magnetic particles in the adsorption of human antibodies under mammalian cell culture conditions

In this work, we systematically evaluated the potential of using boronic acid functionalized magnetic particles in the capturing of human immunoglobulin G under typical mammalian cell culture conditions. For comparison, Protein A coated magnetic particles were also used. The binding pH was found to significantly influence the adsorption isotherms of boronic acid particles with the higher capacities (0.216 g IgG/g support) being observed at pH 7.4. Comparatively, this value was 0.109 g IgG/g support, for Protein A particles under the same conditions. Both particles revealed very fast adsorption kinetics with more than 70% of the maximum binding capacity being achieved in a few seconds. The effect of glucose and lactate, which are known to interact with boronic acid, was evaluated. For glucose, the binding capacity was significantly influenced by the pH and decreased as pH increased. At pH 9.5, a 70% lower binding capacity was observed for glucose concentrations as low as 0.5 g/l. The effect of lactate was less pronounced and almost pH independent reaching at most 20% decrease in binding capacity. Nevertheless, the effect of both molecules was always lower at pH 7.4. The optimization of the elution conditions enabled complete recovery of bound IgG from boronic acid particles using 50mM Tris-HCl, 200 mM sorbitol, 200 mM NaCl at pH 8.5.     

Novel nucleotide‐responsive hydrogels designed from copolymers of boronic acid and cationic units and their applications as a QCM resonator system to nucleotide sensing

Hydrogels comprised of boronic acid monomer (3), cationic monomer (4), and crosslinker monomer (5) were prepared by radical copolymerization. These hydrogels could efficiently bind nucleotides such as AMP and ATP by a cooperative action of the boronic acid‐cis‐diol complexation and the electrostatic interaction between the cationic unit and the phosphate group. The binding processes were conveniently monitored by the swelling and deswelling behaviors of these hydrogels in aqueous solution. For the hydrogel with the specific monomer composition an interesting “charge inversion” was observable: with increasing AMP or ATP concentration, the cation‐rich hydrogel was gradually charge neutralized, once shrunken at the neutral stage, and then swollen again because of the anion‐rich charge state. These nucleotide‐induced swelling and deswelling phenomena were reproduced on the gold surface of a QCM resonator. Therefore, the present system is not only interesting to consider nucleotide‐induced mechanochemical properties, but also applicable as a sensor to the nucleotide detection. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1302–1310, 2000     

荷移分光光度法测定氨酪酸

氨酪酸(am inobutyric acid),化学名称为γ-氨基丁酸。本品在体内与血氨结合生成尿毒排出体外,有降低血氨及促进大脑新陈代谢的作用,能够增强葡萄糖磷酸酯化酶的活性,利于脑细胞功能的恢复。主要用于脑卒中后遗症、脑动脉硬化症、头部外伤后以及一氧化碳中毒所致昏迷的辅助治疗     

Elaboration of poly(ethyleneimine) coated poly( , -lactic acid) particles. Effect of ionic strength on the surface properties and DNA binding capabilities

Poly( , -lactic acid) (PLA)-based particles, obtained by the emulsification–diffusion process, were surface-modified by electrostatic adsorption of poly(ethylenimine) (PEI). The amount of immobilized PEI and the conformation of the polycation at the interface were dependent on the ionic strength of the media. In the absence of salt, or at low ionic strength, the adsorbed amounts of PEI, the surface charge and the critical concentration for coagulation (CCC) of the modified particles were lower than when the adsorption was achieved at elevated ionic strength. Moreover, at low salt concentration, isotherms were of Langmuir type, suggesting the formation of monolayers. The differences in PEI surface conformation had consequences on the DNA binding capacity of the particles, on the plasmid DNA conformation at the interface and on the DNA release in various media. When PEI was adsorbed in a 50 mM phosphate buffer, the amount of bound plasmid and the strength of binding were higher than when PEI was adsorbed in water. From these differences in physico-chemical properties, one can expect differences in transfection or immunization performances of the vectors.     

Spectrophotometric Studies on the Proton Transfer from the Protonated DNA to 5,10,15,20-Tetrakis[4-(trimethyammonio)-phenyl]porphine

Abstract

The interaction of nucleic acids with 5, 10, 15, 20-tetrakis[4-trimethy-ammonio)phenyl]porpine (TAPP) were investigated on the basis of a mechanistic discussion, and a spectrophotometric method for DNAs was accordingly proposed in the present paper. Depending on the acidity of the solution, TAPP can interact with nucleic acids, producing different absorption features. When the pH of the solution is higher than 6.39, TAPP can interact with both DNAs and RNA, giving a new absorption band at 420.3 nm. If the pH is lower than 6.39, however, the interactions with DNAs (but not RNA) can give an absorption band centered at 436.3 nm. It was found that the absorption band at 436.3 nm originates from the proton transfer from the protonated double-stranded structure of DNA to TAPP. At optimal conditions, the absorbance at 436.3 nm is in proportion to the concentration of the DNAs. Calibration curves were linear in the range of 0±3.0 μg.ml^?1 for calf thymus and 0±3.2 μg.ml^?1 for fish sperm DNA. No interference of 4-fold of RNA was found for the determination of DNAs. The limits of determination (3[sgrave]) were 34.6 ng.ml^?1 for calf thymus DNA and 33.2 ng.ml^?1 for fish sperm DNA, respectively. Four synthetic samples were determined with satisfaction.     

从蓖麻油制取10-羟基癸酸的研究

本文报道了从蓖麻油制取10-羟基癸酸的研究,发现了从蓖麻油制取10-羟基癸酸的新的工艺路线和最佳的工艺条件,用氢氧化钠用量、2-辛醇量、反应时间和反应温度四因子三水平L_9(3~4)进行了正交试验,发现最佳工艺条件是:氢氧化钠(g)/蓖麻油(g):1.25:2-辛醇量(g)/蓖麻油(g):2:反应时间:6h;反应温度:180℃。在这一新的工艺路线和最佳工艺条件下,10-羟基癸酸产率可达75％。     

Activity coefficients of hydrochloric acid and ionic interactions in the system HCl-LiCl-H2O from 5 to 45°C

The activity coefficient of HCl (_A) in aqueous mixtures of HCl and LiCl was determined by emf measurements of cells without liquid junction at five values of the total ionic strength, namely 0.1, 0.5, 1.0, 2.0, and 3.0 mol-kg^–1 (m), and at nine temperatures from 5 to 45°C. The ionic strength fraction (_B) of LiCl in the mixtures was varied from 0 to 0.9. The values of log _A were found to vary linearly with the molality of salt, in accordance with the Harned rule, indicating that the properties of these mixtures are determined primarily by binary ionic interactions of the type H-Li and that ternary interactions are not significant even at I=3m. The results also support the suggestion of Robinson that the interaction parameter _H,Li is insensitive to temperature changes when I is less than 2m.On leave 1977–78 from the University of Durban-Westville, South Africa     

Application of chromatography technology in the separation of active alkaloids from Hypecoum leptocarpum and their inhibitory effect on fatty acid synthase

A method that involved the combination of pH‐zone‐refining counter‐current chromatography and semipreparative reversed‐phase liquid chromatography has been established for the preparative separation of alkaloids from Hypecoum leptocarpum. From 1.2 g of crude sample, 31 mg N‐feruloyltyramine, 27 mg oxohydrastinine, 47 mg hydroprotopine, 25 mg leptopidine, and 18 mg hypecocarpine have been obtained. The structure of the new compound, hypecocarpine, is confirmed based on the analysis of spectroscopic data, including NMR, UV, and IR spectroscopy and positive electrospray ionization mass spectrometry. The known chemical structures were characterized on the basis of ¹H and ¹³C NMR spectroscopy. The purities of the five alkaloids are all over 92.7% as determined by high‐performance liquid chromatography. The alkaloids’ cytotoxicity in breast cancer cells is assessed by using a Cell Counting Kit assay and their inhibitory effect on fatty acid synthase expression is assessed by a Western blot assay. These results suggest that leptopidine could suppress growth and induce cytotoxicity in breast cancer cells and that the cytotoxicity of leptopidine may be related to its inhibitory effect on fatty acid synthase expression.     

Studies on the complexes of 4′-substituted benzo-15-crown-5 ligands with sodium picrate and picric acid

Complexes of nine 4'-substituted benzo-15-crown-5 ligands with sodium picrate were prepared. A good linear relationship of the . values from the UV spectra and Hammett [_p - _m] values was observed. Charge transfer complexes of ten 4'-substituted benzo-l5-crown-5 ligands with picric acid were isolated in crystalline form. The color of the complexes depended on the nature of the substituents. All of the complexes were identified by elemental analyses, UV and IR spectra.     

The first contribution of capillary electrophoresis to the study of abiotic origins of homochirality: investigation of the enantioselective adsorption of 3-carboxy adipic acid on minerals

CE with UV detection was used for the first time to determine the enantioselective adsorption of the short-chain tricarboxylic acid, 3-carboxy adipic acid, on minerals as a mean of investigating plausible mechanisms for the origin of biochemical homochirality on Earth. The use of vancomycine as chiral selector in the separation buffer using the partial filling technique enabled the separation of the two enantiomers of this organic acid in about 12 min. Taking into account that this compound has a low absorption of the UV light, and in order to achieve the sensitivity needed to determine the enantiomeric excess of samples of 3-carboxy adipic acid adsorbed on minerals, we applied a strategy consisting of a field-amplified sample stacking together with the use of a bubble capillary and detection at low wavelength (192 nm). This combination enabled an LOD of about 10(-7) M and the determination of the enantiomeric excess of 3-carboxy adipic acid adsorbed on calcite and feldspar mineral samples at subnanomol levels of this acid. Results showed that an enantioselective adsorption of the enantiomers of 3-carboxy adipic acid on minerals took place.     

A DNA electrochemical sensor prepared by electrodepositing zirconia on composite films of single-walled carbon nanotubes and poly(2,6-pyridinedicarboxylic acid), and its application to detection of the PAT gene fragment

Carboxyl group-functionalized single-walled carbon nanotubes (SWNTs) and 2,6-pyridinedicarboxylic acid (PDC) were electropolymerized by cyclic voltammetry on a glassy-carbon electrode (GCE) surface to form composite films (SWNTs/PDC). Zirconia was then electrodeposited on the SWNTs/PDC/GCE from an aqueous electrolyte containing ZrOCl₂ and KCl by cycling the potential between −1.1 V and +0.7 V at a scan rate of 20 mV s⁻¹. DNA probes with a phosphate group at the 5′ end were easily immobilized on the zirconia thin films, because of the strong affinity between zirconia and phosphate groups. The sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS was used for label-free detection of the target DNA by measuring the increase of the electron transfer resistance (R _et) of the electrode surface after the hybridization of the probe DNA with the target DNA. The PAT gene fragment and polymerase chain reaction (PCR) amplification of the NOS gene from transgenically modified beans were satisfactorily detected by use of this DNA electrochemical sensor. The dynamic range of detection of the sensor for the PAT gene fragment was from 1.0 × 10⁻¹¹ to 1.0 × 10⁻⁶ mol L⁻¹ and the detection limit was 1.38 × 10⁻¹² mol L⁻¹.     

Studies on the syntheses of heterocycles from 3-arylsydnone-4-carbohydroximic acid chlorides with N-arylmaleimides, [1,4]naphthoquinone and aromatic amines

3-Arylsydnone-4-carbohydroximic acid chlorides (1) could react with N-arylmaleimides (3a–b) or 2-methyl-N-phenylmale-imide (3c) to give 3-(3-arylsydnon-4-yl)-5-aryl-3a,6a-dihydro-pyrrolo[3,4-d]isoxazole-4,6-diones (4a–h) or 6a-methyl-3-(3-arylsydnon-4-yl)-5-phenyl-3a,6a-dihydro-pyrrolo[3,4-d]isoxazole-4,6-diones (4i–l), respectively. However, 3-(arylsydnon-4-yl)-naphtho[2,3-d]isoxazole-4,9-diones (6a–d) were obtained in good yield by the reaction of carbohydroximic acid chlorides 1 with [1,4]naphthoquinone. Furthermore, 2-(3-arylsydnon-4-yl)benzoxazoles (9a–d) and 2-(3-arylsydnon-4-yl)benzothiazoles (9e–h) were obtained via the reaction of carbohydroximic acid chlorides 1 with ortho-substituted aromatic amines 7a and b.