Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.     

Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil

Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF₆]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF₆]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98–99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58–60°C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF₄]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF₄]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.     

Effect of mobile phase additives on resolution of some nucleic compounds in high performance liquid chromatography

Here we investigate the chromatographic behavior, with reversed-phase high performance liquid chromatography (RP-HPLC) of nucleic compounds (nucleobases, nucleosides, and nucleotides) on a C₁₈ column in several different mobile phase additives, including1-butyl-3-methylimidazolium tetrafuloroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]) ionic liquids, ammonium formate, and potassium phosphate. The effect of the alkyl group length, the imidazolium ring, and the ionic liquid's counterions on retention and resolution of the samples were tested. The results show the potential application of a used buffer system, ion pairing system, and ionic liquid as mobile phase additives in liquid chromatography resolution of nucleic compounds.     

Effect of replacing [NTf2] by [PF6] anion on the [BMIm][NTf2] ionic liquid confined by gold

The effect of replacing bis(trifluoromethylsulphonyl)imide ([NTf₂]) by hexafluorophosphate ([PF₆]) in room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide ([BMIm][NTf₂]) confined between two gold interfaces is herein reported through molecular dynamics simulations using all-atom non-polarisable force-fields. Five systems were studied ranging from pure [BMIm][NTf₂] to pure [BMIm][PF₆], with [PF₆] molar fractions of 0, 0.125, 0.25, 0.375 and 0.5. Special attention was drawn to investigate the impact of the [PF₆] anion on the IL, in particular on the first layers of the liquid in close contact with the solid gold surface.     

Partial uncompetitive inhibition of horseradish peroxidase by a water-miscible ionic liquid [BMIM][MeSO4

The ionic liquid, 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO₄]), was used to investigate the catalytic mechanism of horseradish peroxidase (HRP). The ionic liquid decreased both K_m and k_cat values for the HRP-catalyzed oxidation of guaiacol (2-methoxyphenol) by H₂O₂. These studies imply that [BMIM][MeSO₄] inhibits the enzyme in an uncompetitive manner. The incorporation of substrate stabilization effects measured by a thermodynamic method into the partial uncompetitive inhibition scheme successfully describes HRP-catalysis in the presence of [BMIM][MeSO₄], which participates as the inhibitor. The inhibition constant of the ionic liquid was 0.051 M. The turn-over number of the native HRP was almost 14-times higher than that of the HRP-ionic liquid complex indicating that [BMIM][MeSO₄] does not form a dead-end complex with HRP.     

One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids

Catalytic conversion of un-pretreated Jatropha oil with high-acid value (13.8 mg KOH/g) to biodiesel was studied in ionic liquids (ILs) with metal chlorides. Several commercial ILs were used to catalyze the esterification of oleic acid. It was found that 1-butyl-3-methylimidazolium tosylate ([BMIm][CH₃SO₃]; a Brønsted acidic IL) had the highest catalytic activity with 93% esterification rate for oleic acid at 140 °C but only 12% biodiesel yield at 120 °C. When FeCl₃ was added to [BMIm][CH₃SO₃], a maximum biodiesel yield of 99.7% was achieved at 120 °C. Because metal ions in ILs supplied Lewis acidic sites, and more of the sites could be provided by trivalent metallic ions than those of bivalent ones. It was also found that the catalytic activity with bivalent metallic ions increased with atomic radius. Mixture of [BMIm][CH₃SO₃] and FeCl₃ was easily separated from products for reuse to avoid producing pollutants.     

Strong substrate-stabilizing effect of a water-miscible ionic liquid [BMIM][BF<Subscript>4</Subscript>] in the catalysis of horseradish peroxidase

The effects of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF₄]), on both thermodynamics and kinetic mechanism of the horseradish peroxidase (HRP)-catalyzed oxidation of guaiacol (2-methoxyphenol) by H₂O₂ were investigated. The ionic liquid stabilized the ground state of guaiacol by causing an 8-fold increase of K_m from 3 to 23 mM upon the addition of 25% (v/v) [BMIM][BF₄]. In addition, the effect of [BMIM][BF₄] in decreasing the k_cat value of HRP catalysis was described by a non-competitive inhibition mechanism. The value of the inhibition constant of [BMIM][BF₄] was 2.9 M indicating that the ionic liquid plays the role of a weak non-competitive inhibitor for HRP catalysis.     

A density functional theory study on the interactions between dibenzothiophene and tetrafluoroborate-based ionic liquids

The interactions between dibenzothiophene (DBT) and N-butyl-N-methylimidazolium tetrafluoroborate ([BMIM][BF₄]), N-butyl-N-methylmorpholinium tetrafluoroborate ([Bmmorpholinium][BF₄]), N-butyl-N-methylpiperdinium tetrafluoroborate ([BMPiper][BF₄]), N-butyl-N-methylpyrrolidinium tetrafluoroborate ([BMPyrro][BF₄]), and N-butylpyridinium tetrafluoroborate ([BPY][BF₄]) were investigated using density functional theory approach. Geometric, electron, and topological properties were analyzed using natural bond orbital, atoms in molecules theory, and noncovalent interaction methods in order to understand intermolecular interactions between DBT and ionic liquids. The result shows that hydrogen bond and van der Waals interactions are widespread in all the ionic liquids-DBT systems. Ion-π interactions between DBT and cation or anion are also observed, while π⁺-π interactions are only found in the [BMIM][BF₄]-DBT and [BPY][BF₄]-DBT systems. The order of interaction energy is [BPY][BF4]-DBT > [BMIM][BF₄]-DBT >> [BMPiper][BF₄]-DBT > [BMPyrro][BF₄]-DBT > [BMmorpholinum][BF₄]-DBT. The energies between DBT and the two ionic liquids containing aromatic cations are significantly higher.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Properties of an ionic liquid-tolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">amyloliquefaciens</Emphasis> CMW1 and its extracellular protease

An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF₃SO₃) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF₃SO₃). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF₃SO₃, [BMIM]Cl, [BMIM]CF₃SO₃, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0–12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Thermal Stability and Activity Regain of Horseradish Peroxidase in Aqueous Mixtures of Imidazolium-Based Ionic Liquids

Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 °C in phosphate buffer and 5–25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 °C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5–10% (v/v) [BMIM][BF₄] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83–87 kJ mol⁻¹). After deactivation, enhanced activity regain of the enzyme, up to 70–80% of the initial activity, was found in 25% (v/v) [BMIM][BF₄] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.     

Obtaining high transesterification activity for subtilisin in ionic liquids

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF(6)]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg(-1) h(-1).     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Hydroxynitrile Lyase Catalysis in Ionic Liquid-containing Systems

The cleavage of mandelonitrile catalysed by hydroxynitrile lyases (HNL) from Prunus amygdalus (PaHNL) and Manihot esculenta (MeHNL) proceeded more rapidly in monophasic aqueous media containing 1-propyl-3-methylimidazolium tetrafluoroborate [C₄MIm][BF₄] than in media containing acetonitrile or THF. Both HNLs were much more thermostable in [C₄MIm][BF₄] than in acetonitrile or THF. The addition of each of the four ionic liquids 1-butyl-, 1-pentyl- and 1-hexyl-3-methylimidazolium tetrafluoroborates at 2–6% (v/v in the aqueous phase) increased both the enzyme activity and the product e.e. in the PaHNL-catalysed transcyanation in an aqueous/DIPE biphasic system. However, MeHNL was inactivated by the ionic liquids, as indicated by the decreased reaction rate, substrate conversion and product e.e.     

The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment

Cellulose resource has got much attention as a promising replacement of fossil fuel. The hydrolysis of cellulose is the key step to chemical product and liquid transportation fuel. In this paper a serials of chloride, acetate, and formate based ionic liquids were used as solvents to dissolve cellulose. The cellulose regenerated from ILs was characterized by FTIR and X-ray powder diffraction. From the characterization and analysis, it was found that the original close and compact structure has changed a lot. After enzymatic hydrolysis, different kinds of ionic liquids (ILs) have different yields of the reducing sugar (TRS). They are 100%, 90.72%, and 88.92% from 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1-butyl-3-methylimidazolium formate ([BMIM][HCOO]) respectively after enzymatic hydrolysis at 50 °C for 5 h. The results indicated that the yields and the hydrolysis rates were improved apparently after ILs pretreatment comparing with the untreated substrates.     

Enzyme Catalysed Synthesis in Ambient Temperature Ionic Liquids

The activity of three different lipases, a glycosidase and a protease in ionic liquids has been studied. Ambient temperature ionic liquids are a new class of solvents that are nonvolatile and nonflammable and thus an interesting alternative to classical organic solvents. Monitoring the synthesis of a simple ester, all lipases were found to exhibit both excellent activity and stability in the non-polar ionic liquid 1-butyl-3-methylimidazohum hexaflurophosphate ([bmin][PF₆], 1). Furthermore, β-galactosidase from E. coli and the Subtilisin protease Savinase^TM were both found to exhibit a hydrolytic activity in a 50% aqueous solution of the water-miscible ionic liquid 1-butyl-3-methyhmidazoUum tetra-fluoroborate ([bmin][BF₄], 2) comparable to the activity observed in 50% aqueous solutions of ethanol and acetonitrile.     

Identification of suitable ionic liquids for application in the enzymatic hydrolysis of rutin by an automated screening

An automated method in milliliter scale was developed for the screening of process parameters concerning the hydrolysis of the flavonoid rutin catalyzed by the rhamnosidase activity of naringinase from Penicillium decumbens. Besides the effect of additives such as ionic liquids and low molecular salts, the productivity in a multiple phase system as well as the recyclability of the enzyme in repetitive batches were studied. The hydrophobic ionic liquid (IL) trihexyl(tetradecyl)phosphonium bis(trifluormethylsulfonyl)imide [P(h₃)t][Tf₂N] was identified to combine the most favorable characteristics out of 23 investigated ILs with regard to enzyme compatibility, substrate solubility and enzyme partition coefficient. Also, for the corresponding cations 1-ethyl-3-methylimidazolium [EMIM], 1-butyl-3-methylimidazolium [BMIM], 1-butyl-1-methylpyrrolidinium [BMPL] and 1-octyl-3-methylimidazolium [OMIM], the entity with the [Tf₂N] anion was best tolerated by the naringinase. With increasing IL content, higher space time yields with up to 1.5 g/(L h) for 80% (v/v) [P(h₃)t][Tf₂N] were achieved. Enhanced specific enzyme activity was observed in the presence of Ca²⁺ ions. By addition of [P(h₃)t][Tf₂N] and calcium chloride, the reactive aqueous phase was successfully used in three repetitive batches with full conversion.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.