Mixed Aqueous-organic Solvents Research Articles

Novel Cold-Adapted Recombinant Laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a Green Catalyst in Biotransformation.

Cold-adapted enzymes are useful tools in the organic syntheses conducted in mixed aqueous-organic or non-aqueous solvents due to their molecular flexibility that stabilizes the proteins in low water activity environments. A novel psychrophilic laccase gene from Kabatiella bupleuri, G3 IBMiP, was spliced by Overlap-Extension PCR (OE-PCR) and expressed in Pichia pastoris. Purified recombinant KbLcc1 laccase has an optimal temperature of 30 °C and pH of 3.5, 5.5, 6.0, and 7.0 in the reaction with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, sinapic acid, and syringaldazine, respectively. Moreover, laccase KbLcc1 is highly thermolabile, as it loses 40% of activity after 30 min at 40 °C and is inactivated at 50 °C after the same period of incubation. The new enzyme remained active with 1 mM of Ni2+, Cu2+, Mn2+, and Zn2+ and with 2 mM of Co2+, Ca2+, and Mg2+, but Fe2+ greatly inhibited the laccase activity. Moreover, 1% ethanol had no impact on KbLcc1, although acetone and ethyl acetate decreased the laccase activity. The presence of hexane (40%, v/v) caused a 58% increase in activity. Laccase KbLcc1 could be applied in the decolorization of synthetic dyes and in the biotransformation of ferulic acid to vanillin. After 5 days of reaction at 20 °C, pH 3.5, with 1 mM ABTS as a mediator, the vanillin concentration was 21.9 mg/L and the molar yield of transformation reached 14.39%.

Characterization of transport mechanisms for controlled release polymer membranes using focused ion beam scanning electron microscopy image-based modelling

Mass transport mechanisms of drug release inside a pellet formulation through polymer membrane were investigated with focused ion beam scanning electron microscopy (FIB-SEM). Through careful control of imaging conditions, sub-surface cross sectional images were acquired on two controlled release membrane samples. Image analysis of porosity, pore size distribution, and pore connectivity in 3D provided a matrix of parameters to quantitatively compare samples from different formulations and processing conditions. Image-based computational fluid dynamics simulations were employed to correlate membrane microporosity with flow permeability. Through high resolution FIB-SEM characterization, quantitative microporosity analysis, and mechanistic investigations of flow via image-based simulations, it was found that a 10% difference in the water content of the mixed aqueous-organic solvent used to dissolve polymers had a significant impact on the controlled release performance. This approach provides an analytical avenue to evaluate effects of permeation enhancers, solvents, and other process conditions on controlled release formulations.

Synthesis of a New functionalized phenol and use as ink-free rewriting

(E)-4-((4′-[2,2′:6′,2′′-terpyridin]-4′-yl) phenyl)diazinyl) phenol (a functionalized phenol derivative bearing terpyridine and diazinyl moieties, abbreviated as L) was synthesized to be reused as a new pH-test paper. A Brønsted acid-base reaction results in the deprotonation of L when combining with OH− in aqueous-organic mixed solvents. After absorbing L onto a filter paper and wiping off the solvent, the prepared pH-test paper can suffer a stepwise color change from faint yellow to crimson when exposing to the solutions having pH from 7 to 14. This new pH-test paper exhibits excellent reversibility and reusability with repeatedly water scrubbing. Meanwhile, we examined that the prepared test-papers can be utilized as specific substrates for ink-free writing.

High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Peptide Recovery and Post-translational Modification Analysis in Monoclonal Antibodies and Antibody-Drug Conjugates.

Reversed phase liquid chromatography with mass spectrometry (RPLC-MS) peptide mapping is routinely used for interrogating molecular and structural attributes such as amino acid composition, sequence variants, and post-translational modifications (PTMs) in antibody-derived therapeutics. RPLC has some limitations that often impact the analysis of certain peptides including large hydrophobic peptides, hydrophilic di-/tripeptides and glycopeptides. Capillary zone electrophoresis with mass spectrometry (CZE-MS) has great potential for peptide mapping due to high efficiency and outstanding sensitivity. In this report we demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS for peptide mapping analyses of antibody-drug conjugates (ADCs) and their parent antibodies. This work is based on high-resolution CZE-MS separation recently developed in our group, where a mixed aqueous-organic solvent system containing N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF) was used to improve the separation selectivity. The results described here show several advantages of CZE-MS for the analysis of small hydrophilic di-/tripeptides, large hydrophobic peptides, glycopeptides, and hydrophobic drug-linked peptides.

Inverse thermoresponse of a water-soluble helicene oligomer in aqueous-organic mixed solvent systems

A water-soluble ethynylhelicene (M)-tetramer with TEG groups exhibited the inverse thermoresponse in water/triethylamine/acetone, in which heating induced the formation of double helix and cooling dissociation. In this study, we examined the solvent effect with regard to water content at temperatures between 5 and 40 °C, and showed that the inverse thermoresponse occurred in different aqueous solution systems. In acetone, the oligomer exhibited the ordinary thermoresponse: Cooling induced the formation of double helix and heating dissociation. The increase in water content to approximately 10% caused the switch to the inverse thermoresponse. Further increase inhibited the association, and did not induce the thermoresponse. When the water content was increased to approximately 50%, the inverse thermoresponse appeared as observed previously study. Two types of inverse thermoresponses, termed type I and II, appeared at high and low water contents, respectively.

Solvation and complexation of europium(III) ions in triflate and chloride aqueous-organic solutions by TRLF spectroscopy

Solvation of europium ions in mixed aqueous-organic solvents was studied in the solutions of europium(III) triflate and chloride. Europium ions are preferentially solvated by organic solvent only in water-dimethylsulfoxide systems. In aqueous dimethylformamide and dimethylacetamide preferential hydration in water-deficient region was observed. The total solvation number was proved to decrease from 9 in neat water to 8 in neat dimethylsulfoxide and dimethylformamide and to 7 in neat dimethylacetamide. The formation of chlorocomplexes in neat water and aqueous dimethylacetamide was observed.

Characterization of the Dynamic Equilibrium between Closed and Open Forms of the Benzoxaborole Pharmacophore.

Benzoxaboroles are a class of five-membered hemiboronic acids that recently attracted significant attention as a new pharmacophore on account of their unique structural and physicochemical properties and their ability to interact selectively with biomolecules. Their structural behavior in water and its effect on their physiological properties remain unclear, especially the question of dynamic hydrolytic equilibrium of the oxaborole ring. Herein, we used NMR spectroscopy, in mixed aqueous-organic solvent, to confirm the strong preference for the closed form of benzoxaborole and its six- and seven-membered homologues over the open boronic acid form. Only with the eight-membered homologue does the cyclic form become unfavorable. Using dynamic VT-NMR studies with designed probe compound 20, we demonstrate that the oxaborole ring undergoes rapid hydrolytic ring closing-opening at ambient temperature at a rate of >100 Hz via a mechanism featuring rate-limiting proton-transfer steps. This knowledge can help provide a better understanding of the behavior of benzoxaboroles in biological systems.

Unperturbed dimension, interaction parameters, zeta potential and rheology of sodium alginate in binary solvent mixtures

The intrinsic viscosity [η] of sodium alginate having different molecular weights (high molecular weight (type A), medium molecular weight (type B) and low molecular weight (type C)) is measured at 293–303 K temperature in various mixtures of water (good solvent), acetone (ACE, poor solvent) and water-ethoxy ethanol (EE, poor solvent). The observed result particularly the Huggins constant (K H) values shows a significant variation of cosolvency as a function of solvent composition (ΦACE/ΦEE) and the temperature. Unperturbed dimensions (K θ) under non-theta condition have been calculated using various equations in different water-acetone and water-ethoxy ethanol mixtures. The value of K θ obtained from three different methods of measurements viz., Burchard-Stockmayer and Fixman (BSF), Berry and Inagaki-Suzuki-Kurata (ISK) agree well with each other except in a few compositions of solvents. The molecular extension factor (α n) and actual end-to-end distance (α n K θ) have also been computed herein. Further, the shear thinning nature of the sodium alginate solutions (representative illustration: type A) in water and water-acetone mixed systems has been investigated at increasing strain rate. Results show that the viscous modulus G″ predominates over the elastic modulus G′ and the systems behave as viscoelastic fluids under present conditions. Higher value of G′ in acetone-water mixed solvent system compared to that in pure water undoubtedly indicates enhancement of the elasticity in alginate system in the presence of acetone. Finally, zeta potential measurement demonstrates that the polymeric surface of sodium alginate (representative illustration: type A) is essentially negatively charged and the zeta potential of the polymer is more responsive to the composition of ethoxy ethanol than that of acetone in the aqueous-organic mixed solvents.

Scalable Synthesis of a Nucleoside Phosphoramidate Prodrug Inhibitor of HCV NS5B RdRp: Challenges in the Production of a Diastereomeric Mixture

A scalable process is described for the synthesis of 2′-C-methylguanosine-5′-[2-[(3-hydroxy-2,2-dimethyl-1-oxopropyl)thio]ethyl-N-benzylphosphoramidate], a nucleotide prodrug inhibitor of hepatitis C virus NS5B polymerase. The route features the use of phenylboronic acid to transiently protect the 2′,3′-hydroxyls of 2′-C-methylguanosine under mild conditions. The requirement to produce a 1:1 phosphorus diastereomeric mixture precluded the use of traditional crystallization techniques. High sensitivity of the drug substance to acidic and basic conditions and its preferred solubility in mixed aqueous–organic solvents presented additional processing challenges. The use of reverse phase chromatography for the final purification was eliminated by the development of a dual liquid–liquid extraction protocol, which removed both non-polar and polar impurities whilst maintaining the 1:1 diastereomeric ratio. Ethylene sulfide, a potential genotoxic impurity that was observed at significant levels in the original pro...

Biomimetic peptide-based models of [FeFe]-hydrogenases: utilization of phosphine-containing peptides.

Two synthetic strategies for incorporating diiron analogues of [FeFe]-hydrogenases into short peptides via phosphine functional groups are described. First, utilizing the amine side chain of lysine as an anchor, phosphine carboxylic acids can be coupled via amide formation to resin-bound peptides. Second, artificial, phosphine-containing amino acids can be directly incorporated into peptides via solution phase peptide synthesis. The second approach is demonstrated using three amino acids each with a different phosphine substituent (diphenyl, diisopropyl, and diethyl phosphine). In total, five distinct monophosphine-substituted, diiron model complexes were prepared by reaction of the phosphine-peptides with diiron hexacarbonyl precursors, either (μ-pdt)Fe2(CO)6 or (μ-bdt)Fe2(CO)6 (pdt = propane-1,3-dithiolate, bdt = benzene-1,2-dithiolate). Formation of the complexes was confirmed by UV/Vis, FTIR and (31)P NMR spectroscopy. Electrocatalysis by these complexes is reported in the presence of acetic acid in mixed aqueous-organic solutions. Addition of water results in enhancement of the catalytic rates.

Dependence of the solubility of Cr(III) and Co(III) trisacetylacetonates in mixed aqueous N,N-dimethylformamide solutions depending on solvent composition and temperature

Data on the solubility of cobalt(III) and chromium(III) trisacetylacetonates in water and aqueous-dimethylformamide solutions over a broad range of temperatures and concentrations are presented and the thermodynamic parameters of dissolution are calculated. The inversion point corresponding to the change in the hydration mechanism associated with the transition of the solvent’s structure from an aqueous solution to that of a mixed aqueous-organic solvent is found in dependences of solubility on the molar concentration of the organic component.

Deposition of silver nanoparticles on organically-modified silica in the presence of lignosulfonate

It is shown that the chemical reduction of silver ions by lignosulfonate (LS) in a mixed aqueous–organic solvent produces silver nanoparticles (AgNPs). If, additionally, spherical silica (SiO2) (surface-functionalized with various organic groups) is introduced to the reaction mixture, the LS-stabilized Ag-NPs are deposited on the surface of the silica spheres, forming a SiO2–LS–AgNPs hybrid material. The efficiency of the process is found to depend significantly on both the polarity of the organic solvent and the hydrophobicity of the SiO2-grafted functionalities. The most effective synthesis was in a mixed dimethylformamide–water solvent with octadecylsilane-functionalized SiO2. It is concluded that hydrophobic forces are essential for the successful coupling of LS–AgNPs with the surface of the modified silica. The formation of LS–AgNPs was monitored by UV-Vis spectroscopy, and the properties of the final hybrid materials were determined by EDS, elemental analysis, NIBS, TGA colorimetric analysis and HRTEM techniques. The resulting SiO2–LS–AgNPs hybrids were also used as SERS substrates with Rhodamine 6G as a test molecule.

Dimerization of methylene blue in aqueous and mixed aqueous organic solvent: A spectroscopic study

The dimerization of methylene blue (MB) dye in aqueous and mixed aqueous organic solvents has been investigated spectrophotometrically at 303.15K. The organic solvents chosen for the study include ethylene glycol, 2-methoxy ethanol, 2-ethoxy ethanol, tetrahydrofuran and dioxane. The dimerization constant KD of MB in aqueous as well as the mixed aqueous media has been computed from their UV–visible spectra. Addition of the organic solvent of low dielectric constant resulted into lowering of the KD perhaps due to the overall decrease in dielectric constant of mixed media. However, decrease in KD in the mixed media was not in order of decreasing dielectric constant of the mixed media. In fact, introduction of hydrophobic methyl or ethyl group in ethylene glycol led to an increase in KD in the order 2-ethoxy ethanol>2-methoxy ethanol>ethylene glycol even though the dielectric constant of ethylene glycol is higher than that of methyl or ethyl substituted ethylene glycol. The change in the fraction of monomeric dye (α) in the presence of the mixed aqueous organic solvent as a function of the dye concentration has also been computed. The spectra for the monomer and the dimer species of MB have been re-constructed using the KD values and the results have been analysed in the light of the molecular exciton theory. Both the twist (θ1) and the tilt (θ2) angles between the transition dipoles of the MB molecules in the dimer have been computed. It was observed that the presence of the organic solvent led to decreasing not only the twist but also the tilt angle. The results indicated that the dimer essentially has sandwich geometry with a slight deviation from the exact parallel stacking arrangement of the dye molecular planes highlighting the importance of dispersive forces including the hydrophobic effects in the formation of the dye aggregates.

Dye-surfactant interactions in mixed aqueous-organic solvent media with different hydrophobicity

Dye-surfactant interactions in mixed aqueous-organic solvent media with different hydrophobicity

Protonation equilibria of biologically active ligands in mixed aqueous organic solvents.

The review is mainly concerned with the protonation equilibria of biologically active ligands like amino acids, peptides, DNA constituents, and amino acid esters in nonaqueous media. Equilibrium concentrations of proton-ligand formation as a function of pH were investigated. Also, thermodynamics associated with protonation equilibria were also discussed.

The rate of chloroacetic acid alkaline hydrolysis: Medium effect

Rate constant of chloroacetic acid alkaline hydrolysis in the mixed aqueous-organic solvents depends on the organic co-solvent nucleophilic solvation; however, the effect of the medium polarity on the rate constant is not clear. The rate constant can be reasonably estimated with a multi-parameter equation accounting for the organic co-solvent basicity as well as its density of cohesion energy and molar volume.

Spectral Investigations of Preferential Solvation and Solute–Solvent Interactions of Free Base and Protonated 5,10,15,20-Tetrakis(4-trimethyl-ammonio-phenyl)-porphine Tetratosylate in Aqueous Organic Mixed Solvents

The solvatochromic properties of the free base and the protonated 5,10,15,20-tetrakis(4-trimethyl-ammonio-phenyl)-porphine tetratosylate (TTMAPP) were studied in pure water, methanol, ethanol, 2-propanol, and their corresponding aqueous mixtures. The correlation of the empirical solvent polarity scale (E T) values of TTMAPP with composition of the solvents were analyzed by the solvent exchange model of Bosch and Roses to clarify the preferential solvation of the probe dyes in the binary mixed solvents. The solvation shell composition effects in preferential solvation of the solute dyes were investigated in terms of both solvent–solvent and solute–solvent interactions and also the local mole fraction of each solvent composition was calculated in the cybotactic region of the probe. The effective mole fraction variation may provide significant physicochemical insights in the microscopic and molecular level of interactions between TTMAPP species and the solvent components and, therefore, can be used to interpret the solvent effect on kinetics and thermodynamics of TTMAPP.

Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi

BackgroundHalorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures.ResultsAnalysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2) gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (−5 to 60°C) with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10–20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol.ConclusionThe H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is successful in a perennially cold, hypersaline environment, with relevance to astrobiology.

Critical assessment of the spectroscopic activity assay for monitoring trypsin activity in organic–aqueous solvent

Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous–organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems. However, as reported here, the changing solvent polarity induces bias in the absorbance measurement, lending upward of 35% error in the apparent trypsin activity as the acetonitrile is raised to 70%. Furthermore, time-dependent spectroscopic and mass spectrometric measurements reveal a progressive deactivation of trypsin over a 5- to 10-min period in as little as 30% acetonitrile.

A Priori Prediction of Thermodynamic Properties of Electrolytes in Mixed Aqueous–Organic Solvents to Extreme Temperatures

The unified theory of electrolytes (J. Phys. Chem. B 2009, 113, 2398-2404) for predicting the standard state thermodynamic properties of aqueous electrolytes has been extended to include mixed solvent systems. The solubility of solid sodium chloride in mixed solvents (methanol/water concentration up to 75% w/w) was also measured up to 466 K and pressures near 7 MPa. The present model, together with a simple modification of Pitzer's thermodynamic treatment of aqueous solutions, allows a priori prediction of solubility of electrolytes in aqueous/organic systems to extreme temperatures and pressures. Solubility is predicted for sodium chloride and potassium chloride in mixed solvents (methanol/water, ethanol/water) over a wide range of temperatures and compositions from the extension of the unified theory of electrolytes to mixed solvents. Comparisons indicate good agreement in all cases to well within the uncertainties of the experimental data. The stoichiometric activity coefficients of saturated solution of sodium chloride in methanol/water mixed solvents were calculated up to 473.15 K. The stoichiometric activity coefficients, as a function of temperature at all concentrations (0 ≤ m ≤ m(sat)) and the entire range of mole fraction of methanol, were also calculated up to 473.15 K. The novelty of the present approach is that no additional parameters are required to account for the medium effect.