Efficient three phase partitioning of actinidin from kiwifruit (Actinidia deliciosa) and its characterization.

Three phase partitioning (TPP) method was effectively utilized for the extraction and purification of milk clotting protease (actinidin) from the kiwifruit pulp. The different purification parameters of TPP such as ammonium sulfate saturation, ratio of the crude kiwifruit extract to tert-butanol, and the pH value of extract were optimized. The 40% (w/v) salt saturation having 1.0:0.75 (v/v) ratio of crude kiwifruit extract to tert-butanol at 6.0 pH value exhibited 3.14 purification fold along with 142.27% recovery, and the protease was concentrated exclusively at intermediate phase (IP). This fraction showed milk-clotting activity (MCA), but there was no such activity in lower aqueous phase (AP). The enzyme molecular weight was found to be 24 kDa from Tricine SDS-PAGE analysis. Recovered protease demonstrated greater stability at pH 7.0 and temperature 50 °C. The Vmax and Km values were 121.9 U/ml and 3.2 mg/ml respectively. Its cysteine nature was demonstrated by inhibition studies. This study highlighted that the TPP is an economic and effective method for extraction and purification of actinidin from kiwifruit, and it could be used as a vegetable coagulant for cheesemaking.

Mechanistic Investigation of tert-Butanol's Impact on Biopharmaceutical Formulations: When Experiments Meet Molecular Dynamics.

The use of tert-butyl alcohol for the lyophilization of pharmaceuticals has seen an uptick over the past years. Its advantages include increased solubility of hydrophobic drugs, enhanced product stability, shorter reconstitution time, and decreased processing time. While the mechanisms of protein stabilization exerted by cryo- and lyo-protectants are well known when water is the solvent of choice, little is known for organic solvents. This work investigates the interactions between two model proteins, namely, lactate dehydrogenase and myoglobin, and various excipients (mannitol, sucrose, 2-hydroxypropyl-ß-cyclodextrin and Tween 80) in the presence of tert-butyl alcohol. We thermally characterized mixtures of these components by differential scanning calorimetry and freeze-drying microscopy. We also spectroscopically evaluated the protein recovery after freezing and freeze-drying. We additionally performed molecular dynamics simulations to elucidate the interactions in ternary mixtures of the herein-investigated excipients, tert-butyl alcohol and the proteins. Both experiments and simulations revealed that tert-butyl alcohol had a detrimental impact on the recovery of the two investigated proteins, and no combination of excipients yielded a satisfactory recovery when the organic solvent was present within the formulation. Simulations suggested that the denaturing effect of tert-butyl alcohol was related to its propensity to accumulate in the proximity of the peptide surface, especially near positively charged residues.

Perfluoro-tert-butanol for selective on-resin detritylation: a mild alternative to traditionally used methods.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, Nα-Fmoc-Nε-(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF3)3COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF3)3COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH2Cl2 (3 × 5 min) as well as 5% trichloroacetic acid in CH2Cl2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF3)3COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF3)3COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF3)3COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue.

t-Butanol Enables Dual Functionality of Mannitol: A Cryoprotectant in Frozen Systems and Bulking Agent in Freeze-Dried Formulations.

The effect of tertiary butyl alcohol (TBA) as a cosolvent on the phase behavior of mannitol in frozen and freeze-dried systems was characterized using differential scanning calorimetry (DSC) and X-ray diffractometry (XRD; laboratory and synchrotron sources). Solutions of mannitol (2 and 5% w/w) in TBA-water systems of different compositions (5 to 30% w/w TBA) were characterized, both during cooling and warming using DSC and XRD. At and below the TBA-water eutectic composition (22.5% w/w TBA), mannitol crystallization was completely inhibited in the frozen state, while it crystallized as anhydrous δ-mannitol in the final lyophile. The presence of mannitol did not affect the phase behavior of TBA. The ability of mannitol to serve as a cryoprotectant in frozen solutions, and as a bulking agent in final lyophile was evaluated using human serum albumin (HSA) as a model protein. When HSA in a TBA (5% w/w)-water solution containing mannitol (2% w/w) was freeze-thawed or freeze-dried, there was no evidence of HSA aggregation. Thus, when TBA was used as a cosolvent, mannitol exhibited dual functionality, serving as a cryoprotectant in frozen solutions and as a bulking agent in the final lyophile.

A refined phase diagram of the tert-butanol-water system and implications on lyophilization process optimization of pharmaceuticals.

While water is the solvent of choice for the lyophilization of pharmaceuticals, tert-butyl alcohol (TBA) along with water can confer several advantages including increased solubility of hydrophobic drugs, decreased drying time, improved product stability and reconstitution characteristics. The goal of this work was to generate the phase diagram and determine the eutectic temperature and composition in the "water rich" region (0.0 to 25.0% w/w TBA) of TBA-water mixtures. Solutions of different compositions were frozen and characterized by low temperature differential scanning calorimetry and powder X-ray diffractometry (XRD). The thermal events observed during warming, and their characterization by XRD, enabled the generation of phase boundaries as well as the eutectic temperature and composition. While TBA crystallized as a dihydrate in frozen solutions, on heating, the dihydrate transformed to a heptahydrate. TBA heptahydrate and ice (22.5% w/w TBA) formed a eutectic at ∼-8 °C.

Hydration-Shell Vibrational Spectroscopy.

Hydration-shell vibrational spectroscopy provides an experimental window into solute-induced water structure changes that mediate aqueous folding, binding, and self-assembly. Decomposition of measured Raman and infrared (IR) spectra of aqueous solutions using multivariate curve resolution (MCR) and related methods may be used to obtain solute-correlated spectra revealing solute-induced perturbations of water structure, such as changes in water hydrogen-bond strength, tetrahedral order, and the presence of dangling (non-hydrogen-bonded) OH groups. More generally, vibrational-MCR may be applied to both aqueous and nonaqueous solutions, including multicomponent mixtures, to quantify solvent-mediated interactions between oily, polar, and ionic solutes, in both dilute and crowded fluids. Combining vibrational-MCR with emerging theoretical modeling strategies promises synergetic advances in the predictive understanding of multiscale self-assembly processes of both biological and technological interest.

Hemin-utilizing G-quadruplex DNAzymes are strongly active in organic co-solvents.

The widespread use of organic solvents in industrial processes has focused in recent years on the utility of "green" solvents - those with less harmful environmental, health, and safety properties - such as methanol and formamide. However, protein enzymes, regarded as green catalysts, are often incompatible with organic solvents. Herein, we have explored the oxidative properties of a Fe(III)-heme, or hemin, utilizing catalytic DNA (heme·DNAzyme) in different green solvent-water mixtures. We find that the peroxidase and peroxygenase activities of the heme·DNAzyme are strongly enhanced in 20-30% v/v methanol or formamide, relative to water alone. Protic solvent content of >30% v/v gradually diminishes heme·DNAzyme catalytic activity; however, the heme·DNAzyme is still active in as high as 80% v/v methanol. In contrast to protic solvents, aqueous dimethylformamide solutions largely inhibit heme·DNAzyme activity. In view of the strong catalytic activity of heme·DNAzyme in aqueous methanol, we were able to determine that a 60% v/v methanol-water mixture gives the most optimal yield of the dibenzothiophene sulfoxide (DBTO) oxidation product of petroleum-derived dibenzothiophene (DBT). The high product yield reflects both DNAzyme catalysis and a high substrate availability. Overall, these results emphasize the excellent promise of G-quadruplex forming DNA catalysts in application to "greener" industrial chemistry. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Physicochemical Properties of Solid Phospholipid Particles as a Drug Delivery Platform for Improving Oral Absorption of Poorly Soluble Drugs.

PURPOSE: A novel drug delivery platform, mesoporous phospholipid particle (MPP), is introduced. Its physicochemical properties and ability as a carrier for enhancing oral absorption of poorly soluble drugs are discussed. METHODS: MPP was prepared through freeze-drying a cyclohexane/t-butyl alcohol solution of phosphatidylcholine. Its basic properties were revealed using scanning electron microscopy, x-ray diffraction, thermal analysis, hygroscopicity measurement, and so on. Fenofibrate was loaded to MPP as a poorly soluble model drug, and effect of MPP on the oral absorption behavior was observed. RESULTS: MPP is spherical in shape with a diameter typically in the range of 10-15 µm and a wide surface area that exceeds 10 m2/g. It has a bilayer structure that may accommodate hydrophobic drugs in the acyl chain region. When fenofibrate was loaded in MPP as a model drug, it existed partially in a crystalline state and improvement in the dissolution behavior was achieved in the presence of a surfactant, because of the formation of mixed micelles composed of phospholipids and surfactants in the dissolution media. MPP greatly improved the oral absorption of fenofibrate compared to that of the crystalline drug and its efficacy was almost equivalent to that of an amorphous drug dispersion. CONCLUSION: MPP is a promising option for improving the oral absorption of poorly soluble drugs based on the novel mechanism of dissolution improvement.

Sulfonic Acid Functionalization of Different Zeolites and Their Use as Catalysts in the Microwave-Assisted Etherification of Glycerol with tert-Butyl Alcohol.

The etherification of glycerol with tert-butyl alcohol in the liquid phase, over different sulfonic acid functionalized zeolites, has been studied. The reaction was carried out using microwaves as a way of heating, measured at autogenous pressure and without any solvent. Dealuminated HY and HZSM-5 zeolites by acid treatment were functionalized with two different organosilica precursors: 3-mercaptopropyltrimethoxysilane (M), which incorporates thiol groups, and 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane (C), which incorporates the sulfonic acid groups directly. The thiol groups were oxidized into sulfonic groups employing hydrogen peroxide. The textural and structural properties of the solids were studied by XRD and N2 adsorption-desorption isotherms, whereas the incorporation of the organosilica in the zeolites was studied by TGA and XPS. The novelty functionalization of M gave rise to solids with the highest acidity, and exhibited the highest yields with more substituted ethers (Yh-GTBE = 13%), at 75 °C and 15 min of reaction time. In addition to the acidity, the textural properties of the zeolites played an important role in their activity; HY, with the largest size of the channels, were more active than the HZSM-5.

Producing Radical-Free Hyperpolarized Perfusion Agents for In Vivo Magnetic Resonance Using Spin-Labeled Thermoresponsive Hydrogel.

Dissolution dynamic nuclear polarization (DNP) provides a way to tremendously improve the sensitivity of nuclear magnetic resonance experiments. Once the spins are hyperpolarized by dissolution DNP, the radicals used as polarizing agents become undesirable since their presence is an additional source of nuclear spin relaxation and their toxicity might be an issue. This study demonstrates the feasibility of preparing a hyperpolarized [1-(13) C]2-methylpropan-2-ol (tert-butanol) solution free of persistent radicals by using spin-labeled thermoresponsive hydrophilic polymer networks as polarizing agents. The hyperpolarized (13) C signal can be detected for up to 5 min before the spins fully relax to their thermal equilibrium. This approach extends the applicability of spin-labeled thermoresponsive hydrogel to the dissolution DNP field and highlights its potential as polarizing agent for preparing neat slowly relaxing contrast agents. The hydrogels are especially suited to hyperpolarize deuterated alcohols which can be used for in vivo perfusion imaging.

A sustainable freeze-drying route to porous polysaccharides with tailored hierarchical meso- and macroporosity.

Bio-derived polysaccharide aerogels are of interest for a broad range of applications. To date, these aerogels have been obtained through the time- and solvent-intensive procedure of hydrogel fomation, solvent exchange, and scCO2 drying, which offers little control over meso/macropore distribution. A simpler and more versatile route is developed, using freeze drying to produce highly mesoporous polysaccharide aerogels with various degrees of macroporosity. The hierarchical pore distribution is controlled by addition of different quantities of t-butanol (TBA) to hydrogels before drying. Through a systematic study an interesting relationship between the mesoporosity and t-butanol/water phase diagram is found, linking mesoporosity maxima with eutectic points for all polysaccharides studied (pectin, starch, and alginic acid). Moreover, direct gelation of polysaccharides in aqueous TBA offers additional time savings and the potential for solvent reuse. This finding is a doorway to more accessible polysaccharide aerogels for research and industrial scale production, due to the widespread accessibility of the freeze drying technology and the simplicity of the method.

Cation control of diastereoselectivity in iridium-catalyzed allylic substitutions. Formation of enantioenriched tertiary alcohols and thioethers by allylation of 5H-oxazol-4-ones and 5H-thiazol-4-ones.

We report highly diastereo- and enantioselective allylations of substituted 5H-oxazol-4-ones and 5H-thiazol-4-ones catalyzed by a metallacyclic iridium complex. Enantioselective Ir-catalyzed allylation of substituted 5H-oxazol-4-ones occurs with high diastereoselectivity by employing the corresponding zinc enolates; enantioselective Ir-catalyzed allylation of substituted 5H-thiazol-4-ones occurs with the corresponding magnesium enolates with high diastereoselectivity. The allylation of substituted 5H-oxazol-4-ones provides rapid access to enantioenriched tertiary α-hydroxy acid derivatives unavailable through Mo-catalyzed allylic substitution. The allylation of substituted 5H-thiazol-4-ones provides a novel method to synthesize enantioenriched tertiary thiols and thioethers. The observed cation effect implies a novel method to control the diastereoselectivity in Ir-catalyzed allylic substitution.

Glycerol etherification with TBA: high yield to poly-ethers using a membrane assisted batch reactor.

In this work, a novel approach to obtain high yield to poly-tert-butylglycerolethers by glycerol etherification reaction with tert-butyl alcohol (TBA) is proposed. The limit of this reaction is the production of poly-ethers, which inhibits the formation of poly-ethers potentially usable in the blend with conventional diesel for transportation. The results herein reported demonstrate that the use of a water permselective membrane offers the possibility to shift the equilibrium toward the formation of poly-ethers since the water formed during reaction is continuously and selectively removed from the reaction medium by the recirculation of the gas phase. Using a proper catalyst and optimizing the reaction conditions, in a single experiment, a total glycerol conversion can be reached with a yield to poly-ethers close to 70%, which represents data never before reached using TBA as reactant. The approach here proposed could open up new opportunities for all catalytic reactions affected by water formation.

The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study.

Candida antarctica lipase B (CALB) is an efficient biocatalyst for hydrolysis, esterification, and polymerization reactions. In order to understand how to control enzyme activity and stability we performed a combined experimental and molecular dynamics simulation study of CALB in organic solvents and ionic liquids (ILs). Our results demonstrate that the conformational changes of the active site cavity are directly related to enzyme activity and decrease in the following order: [Bmim][TfO] > tert-butanol > [Bmim][Cl]. The entrance to the cavity is modulated by two isoleucines, ILE-189 and ILE-285, one of which is located on the α-10 helix. The α-10 helix can substantially change its conformation due to specific interactions with solvent molecules. This change is acutely evident in [Bmim][Cl] where interactions of LYS-290 with chlorine anions caused a conformational switch between α-helix and turn. Disruption of the α-10 helix structure results in a narrow cavity entrance and, thus, reduced the activity of CALB in [Bmim][Cl]. Finally, our results show that the electrostatic energy between solvents in this study and CALB is correlated with the structural changes leading to differences in enzyme activity.

Insights into the metalation of benzene and toluene by Schlosser's base: a superbasic cluster comprising PhK, PhLi, and tBuOLi.

The metalation of benzene by Schlosser's base (nBuLi/tBuOK) occurs smoothly in THF at low temperatures to afford a discrete mixed-metal Li2 K4 cluster that contains phenyl anions and tert-butoxide. The aggregate itself exhibits superbasic behavior by metalating toluene. The delocalized benzyl anion obtained this way π bonds to potassium counterions, thereby creating a 2D coordination polymer.

Trimethylamine N-oxide (TMAO) and tert-butyl alcohol (TBA) at hydrophobic interfaces: insights from molecular dynamics simulations.

TMAO, a potent osmolyte, and TBA, a denaturant, have similar molecular architecture but somewhat different chemistry. We employ extensive molecular dynamics simulations to quantify their behavior at vapor-water and octane-water interfaces. We show that interfacial structure-density and orientation-and their dependence on solution concentration are markedly different for the two molecules. TMAO molecules are moderately surface active and adopt orientations with their N-O vector approximately parallel to the aqueous interface. That is, not all methyl groups of TMAO at the interface point away from the water phase. In contrast, TBA molecules act as molecular amphiphiles, are highly surface active, and, at low concentrations, adopt orientations with their methyl groups pointing away and the C-O vector pointing directly into water. The behavior of TMAO at aqueous interfaces is only weakly dependent on its solution concentration, whereas that of TBA depends strongly on concentration. We show that this concentration dependence arises from their different hydrogen bonding capabilities-TMAO can only accept hydrogen bonds from water, whereas TBA can accept (donate) hydrogen bonds from (to) water or other TBA molecules. The ability to self-associate, particularly visible in TBA molecules in the interfacial layer, allows them to sample a broad range of orientations at higher concentrations. In light of the role of TMAO and TBA in biomolecular stability, our results provide a reference with which to compare their behavior near biological interfaces. Also, given the ubiquity of aqueous interfaces in biology, chemistry, and technology, our results may be useful in the design of interfacially active small molecules with the aim to control their orientations and interactions.

reliable Synthesis of Monodisperse Microparticles: Prevention of Oxygen Diffusion and Organic Solvents Using Conformal Polymeric Coating onto Poly(dimethylsiloxane) Micromold.

An effective polymeric thin film deposited by initiated chemical vapor deposition (iCVD) process was presented and its application as a barrier film on the PDMS micromold blocking the penetration of oxygen and organic solvents was investigated. With this barrier film, we were able to synthesize monodisperse polymeric particles of sizes down to 3 µm, which has been reported to be extremely challenging with bare PDMS micromold. The polymeric barrier film on the PDMS micromold enabled this successful synthesis of microparticles by effectively blocking the diffusion of oxygen, which is a well-known radical quencher in radical polymerization, through the PDMS micromold. Furthermore, the iCVD barrier film substantially decreased the penetration of various organic solvents such as acetone, tert-butanol, PDMS oil, and decane as well as organic substances including fluorescent molecules like rhodamine B and fluorescein isothiocyanate (FITC). Therefore, the polymeric barrier film coated on PDMS micromold via iCVD process will broaden the application of PDMS to microfluidic area for the synthesis of smaller microparticles with various organic substances.

Purification, recovery, and characterization of chick pea (Cicer arietinum) ß-galactosidase in single step by three phase partitioning as a rapid and easy technique.

In this study chick pea ß-galactosidase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 60% ammonium sulfate saturation (w/v) with 1:0.5 (v/v) ratio of crude extract:t-butanol at pH 6.8, which gave 10.1 purification fold with 133% recovery of ß-galactosidase. SDS-PAGE analysis showed that protein has two subunits with molecular masses of 48 and 38kDa, respectively. Characterization of enzyme showed that optimal pH of purified enzyme was 2.8 and optimal temperature was 50°C. Enzyme was further characterized by the Arrhenius activation energy and Michael-Menten kinetic constants. Activation energy (Ea) was calculated by using Arrhenius equation and determined to be 15.52kcalmol(-1). Km value of purified enzyme was estimated for the o-nitrophenyl ß-d galactopyranoside (ONPG) substrate as 1.09mM, while its maximum velocity, Vm was 0.90U/mL/min at 37°C. TPP improved substrate affinity of enzyme by the increased flexibility during the partitioning. TPP is simple, easy and economic technique for purification and recovery of ß-galactosidase from chick pea, and has a big potential use for industrial applications.

5-S-lipoylhydroxytyrosol, a multidefense antioxidant featuring a solvent-tunable peroxyl radical-scavenging 3-thio-1,2-dihydroxybenzene motif.

5-S-Lipoylhydroxytyrosol (1), the parent member of a novel group of bioinspired multidefense antioxidants, is shown herein to exhibit potent peroxyl radical scavenging properties that are controlled in a solvent-dependent manner by the sulfur center adjacent to the active o-diphenol moiety. With respect to the parent hydroxytyrosol (HTy), 1 proved to be a more potent inhibitor of model autoxidation processes in a polar solvent (acetonitrile), due to a lower susceptibility to the adverse effects of hydrogen bonding with the solvent. Determination of O-H bond dissociation enthalpies (BDE) in t-butanol by EPR radical equilibration technique consistently indicated a ca. 1.5 kcal/mol lower value for 1 relative to HTy. In good agreement, DFT calculations of the BDEOH using an explicit methanol molecule to mimic solvent effects predicted a 1.2 kcal/mol lower value for 1 relative to HTy. Forcing the geometry of the -S-R group to coplanarity with the aromatic ring resulted in a dramatic decrease in the computed BDEOH values suggesting a potentially higher activity than the reference antioxidant α-tocopherol, depending on geometrical constrains in microheterogeneous environments. These results point to sulfur substitution as an expedient tool to tailor the chain-breaking antioxidant properties of catechol derivatives in a rational and predictable fashion.

Degradation of organic pollutants in wastewater by bicarbonate-activated hydrogen peroxide with a supported cobalt catalyst.

Developing novel technologies to cleanup wastewater has attracted attention for a long while in academic and industrial communities not only for environmental issues but also for recycling water sources. This work demonstrates that bicarbonate-activated H2O2 can be applied as a novel oxidant source in pollutant degradation. Using a supported cobalt catalyst, bicarbonate-activated H2O2 can efficiently degrade various dyes and phenol at ambient temperature. Because the reaction media remains weakly basic during degradation, the cobalt leaching from the solid catalyst has been efficiently avoided and the lifetime of the catalyst can be extended to above 180 h without significant activity loss in a fixed-bed test. Different scavengers, including ascorbic acid, t-butanol, sodium azide, benzoquinone, and tiron, have been tested to identify the active species, which may be involved in pollutant degradation, and it was found that singlet oxygen and the carbonate radical may play a key role in the degradation process.