Activity of α‐Chymotrypsin in Cationic and Nonionic Micellar Media: Ultraviolet and Fluorescence Spectroscopic Approach

α‐Chymotrypsin (α‐CT) activity was measured in aqueous buffer with the following alkyltriphenylphosphonium bromide surfactants in the series cetyl, tetradecyl, and dodecyl as a tail length. For the sake of comparison with mixed micellar investigation on activity of α‐CT, cationic cetyltriphenylphosphonium bromide (CTPB) and nonionic surfactant Triton X‐100, Brij‐56, Brij‐35, Tween 20, and Igepal Co‐210 have been used. The p‐nitrophenyl acetate (PNPA) hydrolysis rate was determined at the surfactant concentration of both cationic and mixed micellar systems by a UV–vis spectrophotometer. The catalytic reaction follows the Michaelis–Menten mechanism, and the catalytic efficiency (k_cat/K_M) was evaluated for both homogeneous and mixed‐micellar media. The maximum catalytic efficiency was observed at 5 mM concentration of CTPB, but the highest catalytic efficiency, 572 M^?1 s^?1, was measured in the presence of mixed micellar (7.5 mM CTPB + 2.5 mM Tween‐20). The fluorescence (FL) spectra showed the differences of α‐CT conformations in the presence of cationic surfactants. The FL results suggest that the influence of cationic surfactant on proteolysis arises from the interaction with the α‐CT. The binding constant, k_sv, of α‐CT with cationic aggregates was determined in the buffer using the Stern–Volmer equation by the fluorescence spectroscopic approach.     

Effects of head group of cationic surfactants on the hydrolysis of p‐nitrophenyl acetate catalyzed by α‐chymotrypsin

The kinetics of hydrolysis of p‐nitrophenyl acetate catalyzed by α‐chymotrypsin (α‐CT) has been studied in the presence of several cationic surfactants having different head groups maintaining the dodecyl hydrophobic residue and bromide counterion. The enzyme activity was tested in the presence of dodecyl trimethylammonium bromide (DTAB), dodecylpyridinium bromide (DPB), dodecyldimethylethanolammonium bromide (DDMEAB), dodecyldiethylethanolammonium bromide (DDEEAB), benzyldimethyldodecylammonium bromide (BDDAB), and dodecyltriphenylphosphonium bromide (DTPB) surfactants. The extent of superactivity depends upon head groups of surfactants. The activity of α‐CT depends on the surfactant concentration and it varies with the surfactant head group dimensions (DTPB > DDEEAB > DTAB > BDDAB > DDMEAB > DPB). For all surfactants, DTPB exhibits highest superactivity. The effects of surfactants on the apparent kinetic parameters like Michaelis constant K_m and the catalytic constant k_cat have been determined. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 377–381, 2009     

Influence of Amine‐Based Cationic Gemini Surfactants on Catalytic Activity of α‐Chymotrypsin

The α‐chymotrypsin activity was tested in aqueous media with the presence of novel cationic amine–based gemini surfactant, with different spacer chain lengths and head group size, and also compared with the cationic cetyltrimethylammonium bromide (CTAB) and cetyltriphenylphosphonium bromide (CTPB) surfactants and aqueous buffer only. The p‐nitrophenyl acetate (PNPA) hydrolysis rate was monitored in the presence of the surfactant concentration at 30°C. Most of these gemini surfactants gave higher catalytic activity as compared to cationic CTAB and CTPB. The highest superactivity was measured in the presence of gemini 16‐12‐16, [dodecanediyl‐1,12‐bis(cetyldimethylammonium bromide)] surfactant at pH 7.5. The catalytic reaction follows the Michaelis–Menten mechanism. The catalytic rate constants, k_cat, show the same profile that the catalytic affinity; K_M being enhanced with increasing space chain length. The results are favorable for considering that the amine‐based gemini surfactant influences more than both the aqueous and cationic micellar media.     

Study of the Enzymatic Activity of Arthrobacter ureafaciens Neuraminidase by Isothermal Titration Calorimetry

Isothermal titration calorimetry was used to determine the enzymatic activity and thermodynamic activation parameters of Arthrobacter ureafaciens sialidase with the sialyl substrates α‐2,3‐, α‐2,6‐sialyllactoses and α‐2,8‐sialic acid dimer. By monitoring the heat released during hydrolysis, the Michaelis constant (K_m), catalytic rate constant (k_cat), activation energy, activation Gibbs energy, enthalpy, and entropy for different monovalent sialyl conjugates were calculated and found to be consistent with those derived by chromatographic or colorimetric assays. The observed decreases in the activation energy and transition entropy of sialyllactoses were larger than the Michaelis activation parameters of lactose‐free di‐sialic acid because of the specific enzyme activity of A. ureafaciens sialidase.     

Laccase behavior in the microenvironment of water core within a biosurfactant‐based reversed micelles system rhamnolipid/n‐hexanol/isooctane/water

Compared with synthetic surfactants (cetyltrimethyl ammonium bromide, sodium bis(2‐ethylhexyl) sulfosuccinate and Tween‐80), the properties of the aqueous core as well as the microenvironment behavior were investigated in water‐in‐oil microemulsions, which are formed by water and biosurfactant rhamnolipid (RL) in the solvent of isooctane/n‐hexanol (1:1, v/v). Besides, as a typical substrate of lignocellulose, guaiacol was used to detect the laccase activity in reversed micelles (RMs). The results were eventually confirmed that RL‐based RM system has higher solubilization ability, more friendly environmental compatibility and milder reaction microenvironment than the others. In this study, triangle phase diagram of surfactant/n‐hexanol/isooctane/water was constructed to analyze the variation of phase behavior between each RM system. For the RL‐based RM system, the effect of the molar ratio of water to surfactant (ω₀) on enzyme hydrolytic activity was also determined to be shown as a bell‐shaped curve and presented a maximum at ω₀ = 19; the O―H stretching vibrations of water in aqueous core was also studied by analyzing the IR spectrum over the region of 3050–3750 cm ^{? 1}. Moreover, kinetic studies showed that the catalytic efficiency of the laccase in RL‐based RM system was lower than in aqueous solution. Nevertheless, the RM system obtained the highest hydrolysis rate at RL concentration of 1.0CMC, which is 0.055 mM. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of the Cationic Surfactant Moiety on the Structure of Water Entrapped in Two Catanionic Reverse Micelles Created from Ionic Liquid‐Like Surfactants

The behavior of water entrapped in reverse micelles (RMs) formed by two catanionic ionic liquid‐like surfactants, benzyl‐n‐hexadecyldimethylammonium 1,4‐bis‐2‐ethylhexylsulfosuccinate (AOT‐BHD) and cetyltrimethylammonium 1,4‐bis‐2‐ethylhexylsulfosuccinate (AOT‐CTA), was investigated by using dynamic (DLS) and static (SLS) light scattering, FTIR, and ¹H NMR spectroscopy techniques. To the best of our knowledge, this is the first report in which AOT‐CTA has been used to create RMs and encapsulate water. DLS and SLS results revealed the formation of RMs in benzene and the interaction of water with the RM interface. From FTIR and ¹H NMR spectroscopy data, a difference in the magnitude of the water–catanionic surfactant interaction at the interface is observed. For the AOT‐BHD RMs, a strong water–surfactant interaction can be invoked whereas for AOT‐CTA this interaction seems to be weaker. Consequently, more water molecules interact with the interface in AOT‐BHD RMs with a completely disrupted hydrogen‐bond network, than in AOT‐CTA RMs in which the water structure is partially preserved. We suggest that the benzyl group present in the BHD⁺ moiety in AOT‐BHD is responsible for the behavior of the catanionic interface in comparison with the interface created in AOT‐CTA. These results show that a simple change in the cationic component in the catanionic surfactant promotes remarkable changes in the RMs interface with interesting consequences, in particular when using the systems as nanoreactors.     

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

Fragment‐based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small‐molecule enzyme activators. Herein, we describe the discovery and characterization of small‐molecule activators of a glycoside hydrolase (a bacterial O‐GlcNAc hydrolase). A ligand‐observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in k_cat/K_M values (k_cat=catalytic rate constant; K_M=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both K_M and V_max values (V_max=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.     

Nanoscale Control Over Interfacial Properties in Mixed Reverse Micelles Formulated by Using Sodium 1,4‐bis‐2‐ethylhexylsulfosuccinate and Tri‐n‐octyl Phosphine Oxide Surfactants

The interfacial properties of pure reverse micelles (RMs) are a consequence of the magnitude and nature of noncovalent interactions between confined water and the surfactant polar head. Addition of a second surfactant to form mixed RMs is expected to influence these interactions and thus affect these properties at the nanoscale level. Herein, pure and mixed RMs stabilized by sodium 1,4‐bis‐2‐ethylhexylsulfosuccinate and tri‐n‐octyl phosphine oxide (TOPO) surfactants in n‐heptane were formulated and studied by varying both the water content and the TOPO mole fraction. The microenvironment generated was sensed by following the solvatochromic behavior of the 1‐methyl‐8‐oxyquinolinium betaine probe and ³¹P NMR spectroscopy. The results reveal unique properties of mixed RMs and we give experimental evidence that free water can be detected in the polar core of the mixed RMs at very low water content. We anticipate that these findings will have an impact on the use of such media as nanoreactors for many types of chemical reactions, such as enzymatic reactions and nanoparticle synthesis.     

One‐Pot Synthesis of 2‐Oxazolines from Ethyl α‐Cyanocinnamate Derivatives with N‐Bromoacetamide

An efficient method for the one‐pot synthesis of 2‐oxazolines from ethyl α‐cyanocinnamate derivatives with N‐bromoacetamide in the presence of K₃PO₄ has been developed. The reaction performed smoothly and cleanly to give 2‐oxazolines in good to excellent yields (up to 98%) within 4.5 h in acetone at room temperature without protection of inert gases. A total of 13 examples have been investigated. A possible nucleophilic addition reaction mechanism is proposed.     

Kinetic Study of Peroxidase‐Catalyzed Coupling of Benzene‐1,4‐diamine and N‐(2‐Aminoethyl)naphthalen‐1‐amine: Development of Micromolar Hydrogen Peroxide Reaction System

A novel chromogenic method to measure the peroxidase activity using para‐phenylenediamine dihydrochloride (=benzene‐1,4‐diamine hydrochloride; PPDD) and N‐(1‐naphthyl)ethylenediamine dihydrochloride (=N‐(2‐aminoethyl)naphthalen‐1‐amine; NEDA) is presented. The PPDD entraps the free radical and gets oxidized to electrophilic diimine, which couples with NEDA to give an intense red‐colored chromogenic species with maximum absorbance at 490 nm. This assay was adopted for the quantification of H₂O₂ between 20 and 160 μM . Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 4.47×10⁴ M ^?1 min^?1 and 3.38×10^?4 min^?1, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the co‐substrates were 0.0245×10³ min^?1 and 0.0445 μM ^?1 min^?1, respectively. The chromogenic coupling reaction has a minimum interference from the reducing substances such as ascorbic acid, L ‐cystein, citric acid, and oxalic acid. The method being simple, rapid, precise, and sensitive, its applicability has been tested in the crude vegetable extracts that showed peroxidase activity.     

Study of the reaction Fe(CN)5(4‐CNpy)3− + S2O82− in aqueous salt and micellar solutions

The reaction Fe(CN)₅(4‐CNpy)³⁻ + S₂O₈²⁻ (4‐CNpy=4‐cyanopyridine) was studied in aqueous salt solutions in the presence of several electrolytes as well as in anionic, cationic, and nonionic surfactant solutions. In aqueous salt solutions the noncoulombic interactions seem to be important in determining the positive salt effects observed. The salting effects are influencing the activity coefficients of any participant in the reaction, including those ion pairs which can be formed between the anionic reagents and the cations which come from the added salts. The changes in surfactant concentration in anionic and nonionic surfactant solutions do not affect the reaction rate, which is similar to that in pure water at the same ionic strength. In cationic micellar solutions an increase in the rate constant compared to that in pure water is found; the reaction rate decreasing when the surfactant concentration increases. The kinetic trends can be explained assuming that the reagents are totally bound to the micelles and, therefore, an increase in the surfactant concentration results in a decrease in the reagent concentrations at the micellar phase and thus in a decrease in the observed rate constant. © 1999 John Wiley & Sons, Inc. Int J Chem Kinet: 31: 229–235, 1999     

Reactivity Tracers of the Hydrolysis of Fe(II)‐Bis(salicylidene) Complexes: Initial–Transition States Analysis and Enhanced Impact of Tensides on the Kinetic Trends

The kinetics of the acid hydrolysis reaction of Fe(II)‐bis(salicylidene) complexes were followed under pseudo–first‐order conditions ([H⁺] >> [complex]) at 298 K. The ligands of the studied azomethine complexes were derived from the condensation of salicylaldehyde with different five α‐amino acids. The hydrolysis reactions were studied in acidic medium at different ratios (v/v) of aqua–organic mixtures. The decrease in the dielectric constant values of the reaction mixture enhances the reactivity of the reaction. The transfer chemical potentials of the initial and transition states (IS–TS) from water into mixed solvents were determined from the solubility measurements combined with the kinetic data. Nonlinear plots of logk_obs versus 1/D (the reciprocal of the dielectric constant) suggest the influence of the solvation of IS–TS on the reaction reactivity. Furthermore, the acid hydrolysis reactions were screened in the presence of different concentrations of cationic and anionic tensides. The addition of surfactants to the reaction mixture accelerates the reaction reactivity. The obtained kinetic data were used to determine the values of δ_mΔG^# (the change in the activation barrier) for the studied complexes when transferred from “water to various ratios (v/v) of water–co‐organic binary mixtures” and from “water to water containing different [surfactant].” It was found that the reactivity of the acid hydrolysis reaction was controlled by the hydrophobicity of the studied chelates.     

Enantio‐ and Site‐Selective α‐Fluorination of N‐Acyl 3,5‐Dimethylpyrazoles Catalyzed by Chiral π–CuII Complexes

Catalytic enantioselective α‐fluorination reactions of carbonyl compounds are among the most powerful and efficient synthetic methods for constructing optically active α‐fluorinated carbonyl compounds. Nevertheless, α‐fluorination of α‐nonbranched carboxylic acid derivatives is still a big challenge because of relatively high pK_a values of their α‐hydrogen atoms and difficulty of subsequent synthetic transformation without epimerization. Herein we show that chiral copper(II) complexes of 3‐(2‐naphthyl)‐l ‐alanine‐derived amides are highly effective catalysts for the enantio‐ and site‐selective α‐fluorination of N‐(α‐arylacetyl) and N‐(α‐alkylacetyl) 3,5‐dimethylpyrazoles. The substrate scope of the transformation is very broad (25 examples including a quaternary α‐fluorinated α‐amino acid derivative). α‐Fluorinated products were converted into the corresponding esters, secondary amides, tertiary amides, ketones, and alcohols with almost no epimerization in high yield.     

Anchored Mediator Enabling Shuttle‐Free Redox Mediation in Lithium‐Oxygen Batteries

Redox mediators (RMs) are considered an effective countermeasure to reduce the large polarization in lithium‐oxygen batteries. Nevertheless, achieving sufficient enhancement of the cyclability is limited by the trade‐offs of freely mobile RMs, which are beneficial for charge transport but also trigger the shuttling phenomenon. Here, we successfully decoupled the charge‐carrying redox property of RMs and shuttling phenomenon by anchoring the RMs in polymer form, where physical RM migration was replaced by charge transfer along polymer chains. Using PTMA (poly(2,2,6,6‐tetramethyl‐1‐piperidinyloxy‐4‐yl methacrylate)) as a polymer model system based on the well‐known RM tetramethylpiperidinyloxyl (TEMPO), it is demonstrated that PTMA can function as stationary RM, preserving the redox activity of TEMPO. The efficiency of RM‐mediated Li₂O₂ decomposition remains remarkably stable without the consumption of oxidized RMs or degradation of the lithium anode, resulting in an improved performance of the lithium‐oxygen cell.     

Interfacially Cross‐Linked Reverse Micelles as Soluble Support for Palladium Nanoparticle Catalysts

Reverse micelles (RM) were formed in heptane/CHCl₃ with a surfactant carrying the triallylammonium (=triprop‐2‐en‐1‐ylammonium) head group (Scheme). Photo‐cross‐linking with dithiothreitol (=rel‐(2R,3R)‐1,4‐dimercaptobutane‐2,3‐diol; DTT) captured the RMs and afforded organic, soluble nanoparticles in a one‐step reaction. Similar to dendrimers, the cross‐linked reverse micelles could encapsulate palladium nanoparticles within their hydrophilic cores and protect them in catalytic reactions. Good to excellent yields were obtained in the Heck coupling of a range of alkyl acrylates (=alkyl prop‐2‐enoates) and iodobenzenes (Tables 1 and 2). The catalytic activity of the palladium nanoparticles was maintained in several repeated runs.     

Expression,Purification and Characterization of Human α‐l‐Fucosidase

α‐l ‐Fucosidases (EC 3.2.1.51) are exo‐glycosidases. On the basis of the multi‐alignment of amino acid sequence, α‐l ‐fucosidases were classified into two families of glycoside hydrolases, GH‐29 and GH‐95. They are responsible for the removal of l ‐fucosyl residues from the non‐reducing end of glycoconjugates. Deficiency of α‐l ‐fucosidase results in Fucosidosis due to the accumulation of fucose‐containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of α‐l ‐fucosidases may relate to this abnormal cell physiology. Although the gene of human α‐l ‐fucosidase (h‐fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human α‐l ‐fucosidases (h‐Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion‐exchange and gel‐filtration chromatographic purification, the recombinant h‐Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS‐PAGE (～50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h‐Fuc was stable up to 55 °C with incubation at pH 6.8 for 2 h; the optimum temperature for h‐Fuc is approximately 55 °C. The enzyme was stable at pH 2.5–7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K_m and k_cat values of the recombinant h‐Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s^?1, respectively. The study of pH‐dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h‐Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h‐Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two‐step, double displacement mechanism.     

Development of a fast and efficient CE enzyme assay for the characterization and inhibition studies of α‐glucosidase inhibitors

The inhibition of the α‐glucosidase enzyme plays an important role in the treatment of diabetes mellitus. We have established a highly sensitive, fast, and convenient CE method for the characterization of the enzyme and inhibition studies of α‐glucosidase inhibitors. The separation conditions were optimized; the pH value and concentration of the borate‐based separation buffer were optimized in order to achieve baseline separation of p‐nitrophenyl‐α‐d ‐glucopyranoside and p‐nitrophenolate. The optimized method using 25 mM tetraborate buffer, pH 9.5, was evaluated in terms of repeatability, LOD, LOQ, and linearity. The LOD and LOQ were 0.32 and 1.32 μM for p‐nitrophenyl‐α‐d ‐glucopyranoside and 0.83 and 3.42 μM for p‐nitrophenolate, respectively. The value of the Michaelis–Menten constant (K_m) determined for the enzyme is 0.61 mM, which is in good agreement with the reported data. The RSDs (n = 6) for the migration time was 0.67 and 1.83% for substrate and product, respectively. In the newly established CE method, the separation of the reaction analytes was completed in <4 min. The developed CE method is rapid and simple for measuring enzyme kinetics and for assaying inhibitors.     

Aminated PCL‐based copolymers by chemical modification of poly(α‐iodo‐ε‐caprolactone‐co‐ε‐caprolactone)

A novel method is proposed to access to new poly(α‐amino‐ε‐caprolactone‐co‐ε‐caprolactone) using poly(α‐iodo‐ε‐caprolactone‐co‐ε‐caprolactone) as polymeric substrate. First, ring‐opening (co)polymerizations of α‐iodo‐ε‐caprolactone (αIεCL) with ε‐caprolactone (εCL) are performed using tin 2‐ethylhexanoate (Sn(Oct)₂) as catalyst. (Co)polymers are fully characterized by ¹H NMR, ¹³C NMR, FTIR, SEC, DSC, and TGA. Then, these iodinated polyesters are used as polymeric substrates to access to poly(α‐amino‐ε‐caprolactone‐co‐ε‐caprolactone) by two different strategies. The first one is the reaction of poly(αIεCL‐co‐εCL) with ammonia, the second one is the reduction of poly(αN₃εCL‐co‐εCL) by hydrogenolysis. This poly(α‐amino‐ε‐caprolactone‐co‐ε‐caprolactone) (F_αNH2εCL < 0.1) opens the way to new cationic and water‐soluble PCL‐based degradable polyesters. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6104–6115, 2009     

Self‐assembled three‐dimensional flower‐like α‐Fe2O3 nanostructures and their application in catalysis

Three‐dimensional flower‐like α‐Fe₂O₃ nanostructures have been successfully synthesized by a simple surfactant‐free environmental friendly solvolthermal process. The as‐prepared products were investigated by X‐ray powder diffraction, transmission electron microscopy, and field emission scanning electron microscopy. By adjusting the synthetic parameters, the shape of the α‐Fe₂O₃ nanostructures can be controlled. The three‐dimensional flower‐like α‐Fe₂O₃ nanostructures were found to be highly active as catalysts for phenol alkylation. The effects of various parameters, such as reaction temperature, reaction time and the amount of catalyst, were studied. The catalyst was stable and could be reused three times in normal atmosphere without suffering appreciable loss in catalytic activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Palladium(II)‐catalyzed cyclization of W‐(2′,4′‐dienyl)‐2‐alkynamides to α‐alkylidene‐γ‐butyrolactams

In the presence of CuCl₂, N‐(2′, 4′‐dienyl)‐2‐alkynamides can be converted to α‐alkylidene‐σ‐butyrolactams under the catalysis of palladium(II). In this reaction, CuCl₂ is used to oxidize Pd(0) to regenerate Pd(II), or the carbon‐palladium bond is quenched by the oxidative cleavage reaction of CuCl₂.