Biphasic Reaction Medium Research Articles

Chemoenzymatic synthesis of the chiral side-chain of statins: application of an alcohol dehydrogenase catalysed ketone reduction on a large scale

The chemoenzymatic synthesis of the tert-butyl (S)-6-chloro-5-hydroxy-3-ketohexanoate is described. Our approach relies on a highly regio- and enantioselective reduction of a beta,delta-diketohexanoate ester catalysed by NADP(H)-dependent alcohol dehydrogenase of Lactobacillus brevis (LBADH). A detailed description of the scale-up of the enzymatic synthesis of the hydroxyketo ester is given which includes a scale-up of the substrate synthesis as well, i.e. the preparation of diketo ester on a 100 g scale. Furthermore, studies directed towards improving the co-catalyst [NADP(H)] consumption of the enzymatic key step by kinetic studies and application of a biphasic reaction medium were performed.

Enantioselective CC Bond Ligation Using Recombinant Escherichia coli‐Whole‐Cell Biocatalysts

AbstractThiamine diphosphate (ThDP)‐dependent enzymes like benzaldehyde lyase from Pseudomonas fluorescens (BAL) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are versatile biocatalysts for the CC bond ligation of aldehydes to form enantiomerically pure 2‐hydroxy ketones. However, the large‐scale application of this enzyme class is often restricted by the required external addition of the expensive cofactor ThDP, as well as by the common use of dimethyl sulfoxide (DMSO) as a cosolvent, which leads to problems during the work‐up procedure. In the present paper we demonstrate that the addition of the excess cofactors, ThDP and magnesium ions (Mg2+), is not required when BAL or BFD are used in Escherichia coli resting cells. Furthermore, the combination of these resting cells with a biphasic reaction medium [methyl tert‐butyl ether (MTBE)/aqueous buffer] allows an increase of the substrate concentration up to 1 M, and an efficient extractive work‐up. As a practical example, e.g., the synthesis of (R)‐2‐hydroxy‐3,3‐dimethoxy‐phenylpropanone from benzaldehyde and 2,2‐dimethoxyacetaldehyde was optimized, achieving an isolated yield of 78 %, and an enantiomeric excess of 98 % ee in 24 h when operating at a substrate concentration of 0.4 M. The described reaction system in a biphasic medium is suitable for a wide range of aldehydes as substrates. The biphasic reaction medium minimizes also the formation of by‐products, which were observed when this reaction was performed in the conventional DMSO/buffer system.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Catalytic Carbon−Hydrogen Bond Functionalization in an Ionic Liquid Medium

The functionalization of unreactive C−H bonds of several hydrocarbons has been achieved at room temperature by the formal insertion of carbene/CHCO2Et units (from ethyl diazoacetate) into such bonds, using copper-based catalysts in a biphasic reaction medium (ionic liquid-hydrocarbon). Catalyst and product separation has been carried out easily, and the recycle of the catalyst for five cycles has been achieved without any loss of catalytic activity.

Kinetic modeling of enzymatic ethyl oleate synthesis carried out in biphasic systems

This work presents a new approach for the kinetic modeling of solvent free ethyl oleate synthesis, catalyzed by an enzymatic catalyst. The biocatalyst used was the lipase from Candida antarctica B immobilized onto chitosan powder. Thermodynamic calculations revealed that the addition of water to hydrate the biocatalyst, or the production of water during esterification leads to a biphasic reaction medium, due to the formation of a second aqueous phase. A rigorous reactor model is proposed to calculate compositions over time, and to fit kinetic parameters, considering mass transfer and chemical reaction simultaneously. The biphasic model presented, which explicitly considers the presence of a second phase, is compared with the traditional monophasic approach that ignores the biphasic nature of reaction medium. The best-fitted monophasic model is not able to represent experimental data. On the other hand, the biphasic model allows to accurately fit experimental data, indicating the importance of modeling two-phase systems properly.

Oxidation of mono- and bicyclic aromatic compounds with hydrogen peroxide catalyzed by titanium silicalites TS-1 and TS-1B

Abstract The hydroxylation of a series of oxygenated aromatic compounds (phenols and ethers) with hydrogen peroxide, catalyzed by titanium silicalite TS-1 and a new catalyst TS-1B, was investigated operating in a biphasic reaction medium. In several cases, the reaction afforded phenols with fairly good selectivity. TS-1B is often a better catalyst than the conventional TS-1, displaying higher selectivity on hydrogen peroxide and also higher shape selectivity. So it appears to have some potential in the direct synthesis of selected phenols, in alternative to the conventional multi-step methods.

Epoxide Hydrolase‐Catalysed Kinetic Resolution of a Spiroepoxide, a Key Building Block of Various 11‐Heterosteroids

AbstractTwo microbial epoxide hydrolases – i.e., Aspergillus niger (AnEH) and Rhodococcus erythropolis (the so‐called “Limonene EH”: LEH) were used to achieve, for the first time, the biocatalysed hydrolytic kinetic resolution (BHKR) of spiroepoxide rac‐1. This compound is a strategic key building block allowing the synthesis of 11‐heterosteroids. Interestingly enough, the two enzymes exhibited opposite and therefore complementary enantioselectivity allowing us to isolate the residual (R,R)‐1 (from AnEH) and the residual (S,S)‐1 (from LEH) in nearly enantiopure forms (>98 %). Their absolute configurations were determined by X‐ray crystallography. An opposite regioselectivity of the oxirane ring opening for both enantiomers of substrate 1, determined using H218O labelling and chiral GC‐MS analysis, was also observed, corresponding to an attack at the less substituted carbon atom using AnEH, and at the most substituted carbon atom using LEH. A chemical process‐improving methodology was also developed. This allowed us to obtain both enantiomers of the substrate in high enantiomeric purity (99 %) and optimised quantity. In the case of the AnEH, the use of a biphasic (water/isooctane) reaction medium allowed us to increase the global substrate concentration up to 200 g/ L. The preparation of both enantiomers of 1 clearly paves the way to the preparative scale synthesis and biochemical evaluation of the corresponding 11‐heterosteroid enantiomers.

Maximise Equilibrium Conversion in Biphasic Catalysed Reactions: How to Obtain Reliable Data for Equilibrium Constants?

AbstractFor the prediction and optimisation of the equilibrium conversion in biphasic catalysed reactions, the equilibrium constant of the desired reaction and the partition coefficients of all reactants have to be known. Within this contribution we have examined the alcohol dehydrogenase‐catalysed reduction of several linear and aromatic ketones in biphasic reaction media with respect to equilibrium conversion. In this example, the equilibrium constant can be expressed in terms of differences in oxidation‐reduction potentials ΔE0. However, for a large variety of organic compounds, these data are quite rare in the literature. To overcome this lack of data, we have utilised methods of computational chemistry to calculate data for the Gibbs free energy ΔGR leading to the equilibrium constants of a homologous series of linear ketones. To obtain comparable data for the reduction of substituted acetophenone derivatives, the Hammett relation leads to the necessary equilibrium constants. Furthermore, we compare the equilibrium conversions of a set of cofactor regeneration methods for the alcohol dehydrogenase‐catalysed reductions. These results lead to a time‐saving experimental design for the enantioselective reduction of 2‐octanone to (R)‐2‐octanol on a preparative scale utilising biphasic reaction conditions.

Preparative asymmetric reduction of ketones in a biphasic medium with an ( S)-alcohol dehydrogenase under in situ-cofactor-recycling with a formate dehydrogenase

The substrate range of a novel recombinant ( S)-alcohol dehydrogenase from Rhodococcus erythropolis is described. In addition, an enzyme-compatible biphasic reaction medium for the asymmetric biocatalytic reduction of ketones with in situ-cofactor regeneration has been developed. Thus, reductions of poorly water soluble ketones in the presence of the alcohol dehydrogenase from R. erythropolis and a formate dehydrogenase from Candida boidinii can be carried out at higher substrate concentrations of 10–200 mM. The resulting ( S)-alcohols were formed with moderate to good conversion rates, and with up to >99% ee.

Ligand effect on the iron-catalysed biphasic oxidation of aromatic hydrocarbons by hydrogen peroxide

Abstract The hydroxylation of benzene and toluene with hydrogen peroxide, catalysed by iron complexes with nitrogen ligands in a biphasic reaction medium, was investigated. After testing a series of pyridine and pyrazinecarboxylic acid derivatives, it was found that the ligand markedly affects the catalyst efficiency and the selectivity based on the oxidant. In the case of toluene, the ligand has a tuning effect on the catalyst activity, driving the oxidation preferentially to the ring or to the methyl group. The complex with pyrazine-3-carboxylic acid N -oxide was selected as the most efficient catalyst and appears to be a promising tool for the direct synthesis of phenols. The mechanism of the aromatic hydroxylation is also discussed, in comparison with the conventional Fenton’s reagent.

Direct synthesis of phenols by iron-catalyzed biphasic oxidation of aromatic hydrocarbons with hydrogen peroxide

The hydroxylation of a series of aromatic hydrocarbons with hydrogen peroxide, catalyzed by iron complexes with pyrazine-3-carboxylic acid N-oxide, was investigated, operating in a biphasic reaction medium.

Quinone-mediated synthesis of hydrogen peroxide from carbon monoxide, water and oxygen

An improved method for the synthesis of hydrogen peroxide from carbon monoxide, water and oxygen catalyzed by palladium complexes in presence of a quinone co-catalyst is described.Even if the reaction balance is formally the same, the mechanism of formation of hydrogen peroxide in the new process is totally different to that proposed for the reaction carried out in absence of quinones. Using 1,10-phenanthroline as a palladium ligand in a water/organic biphasic reaction medium, a dramatic enhancement of the catalyst stability was obtained operating in presence of 1,4-naphthoquinone, which prevented the formation of inactive binuclear palladium species. Additional improvements, with respect to the reaction carried out in absence of quinone, are the use of cheaper palladium ligands and the safer process conditions, since air can be used as a reagent instead of pure oxygen.

The nickel-substituted quasi-Wells-Dawson-type polyfluoroxometalate,

A series of transition metal substituted polyfluorooxometalates (PFOM) [M(L)H2F6NaW17)55]q-, M= Zn2+ , Co2+, Mn2+, Fc2+, Ru2+, Ni2+ and V5+ and L=H2O, O2-, of quasi-Wells-Dawson structure, was synthesized. In the series prepared, only the nickel-substituted polyfluorooxometalate was capable of catalytic activation of hydrogen peroxide in biphasic reaction media, the reaction leading mainly to the selective epoxidation of alkenes and alkenols. The manganese-, cobalt-, ruthenium-, iron-, vanadium-, and zinc-substituted polyfluorooxometalates were catalytically inactive, although, except for the zinc polyfluorooxometalate, very significant catalase activity was observed. Oxidation of thianthrene showed that sulfoxides were oxidized more easily than sulfides. Kinetic profiles of cyclooctene epoxidation showed that the reaction was zero order in both cyclooctene and hydrogen peroxide. Hydrogen peroxide was consumed at a rate 40% higher than the rate of epoxidation of cyclooctene. The reaction appears to proceed through an intermediate peroxo/hydroperoxo species that was observed in the IR spectrum. Atomic absorption, IR and 19F NMR spectroscopy indicated that the [Ni(H2O)H2F6NaW17O55]9- compound was stable under reaction conditions.

O‐etherification between glycerol and glycerol monooleate—demonstration of formation of diglycerol monooleate and triglycerol monooleate by fast atom bombardment‐mass spectroscopy and 13C nuclear magnetic resonance

AbstractTo prepare glycerol monooleate, we reacted glycerol with oleic acid in the presence of a K1411 sulfonic acid resin in the H+ form. We utilized glycerol monooleate as an emulsifying agent, as it is a nonionic surfactant that is compatible with the initially biphasic reaction medium. If the expected product does not form, small amounts of a highly polar compound are produced. Both diglycerol monooleate and triglycerol monooleate were isolated by preparative chromatography on a silica column (hexane/chloroform/acetone, 25∶50∶50), and were formally identified by fast atom bombardment (FAB)‐mass spectroscopy in positive mode. The products were polyglycerol monoesters. The nature of the products was confirmed by the use of different FAB matrices with or without cationization by alkali metals. The structure of the product was then confirmed by 13C nuclear magnetic resonance. we can now propose a new method of polyglycerol ester preparation that yields interesting nonionic emulsifying agents.

Peroxometalate Catalyzed Oxidations with Hydrogen Peroxide in Biphasic Reaction Media: Reactions in Inverse Emulsions

Peroxometalate Catalyzed Oxidations with Hydrogen Peroxide in Biphasic Reaction Media: Reactions in Inverse Emulsions

Bio-oxidation of aliphatic and aromatic high molecular weight alcohols by Pichia pastoris alcohol oxidase

The alcohol-oxidase-mediated oxidation of hexanol to hexanal was conducted by whole cells of Pichia pastoris in a biphasic reaction medium consisting of 3% water and 97% (v/v) water-saturated hexane. At substrate levels of ca. 10 g/l, hexanal was produced at a rate of 0.2 g/g cell dry wt. per hour with product yields and carbon recoveries of 96% or greater. Although the substrate range of P. pastoris alcohol oxidase has been documented as C1–C5 aliphatic alcohols and benzyl alcohol, the use of a biphasic organic reaction medium showed that this enzyme can also oxidize higher molecular weight aliphatic alcohols of C6–C11, as well as the aromatic alcohols phenethyl alcohol and 3-phenyl-1-propanol. The ability of alcohol oxidase to oxidize low-water-soluble alcohols greatly extends the utility of this enzyme.

Synthesis of U‐14C‐labelled 5‐hydroxymethyl‐2‐furaldehyde

AbstractUniformally 14C‐labelled 5‐hydroxymethyl‐2‐furaldehyde was synthesized by dehydration of D‐[U‐14C]fructose on an H+ ion exchange resin and a water isobutylmethyl ketone biphasic liquid reaction medium. The optimal reaction time was 45min, and after HPLC purification the radiochemical yield was 50%. The whole process took 2 hours.

Synthesis by enzymic catalysis II. Amino acid polymerisation

AbstractAn optimised method for the enzymic oligomerisation of methyl L‐tyrosinate is reported. The effects of the following parameters were studied: the nature of the enzyme, pH, substrate/enzyme ratio and the use of miscible solvents or biphasic media. Optimal conditions were achieved using papain, in an aqueous buffer at pH 5 with a substrate/enzyme ratio of 2.88 × 103 (mole). A tyrosine heptapeptide with a free carboxy terminal was prepared from the tyrosine methyl ester. Using a biphasic reaction medium, the final product had the same structure except that the C‐terminal was in the form of the methyl ester. A semi‐continuous reaction with repeated addition of the ester was required to maintain an adequate substrate/enzyme ratio. Several catalytic cycles could thus be achieved, producing considerable quantities of the oligomers with a limited amount of enzyme. Stereospecificity was demonstrated using the D, L derivative as substrate. Only the L‐tyrosine oligomer was produced, leaving the D‐ester in solution.