Raphael Bar

Microbial transformations in a cyclodextrin medium. Part 2. Reduction of androstenedione to testosterone by Saccharomyces cerevisiae

SummaryAn intensive and systematic investigation of the reductive transformation of androstenedione (AD) to testosterone by Saccharomyces cerevisiae was undertaken in the presence of natural and chemically modified cyclodextrins (CD). The bioconversion was significantly larger in the presence of β- and ψ-CD and hydroxypropyl-β-CD b but only slight in the presence of α-CD and dimethyl- and trimethyl-β-CD. The performance of the various cyclodextrin media was interpreted in the light of the measured phase solubility diagrams of AD. Further investigation focused on biotransformation of the β-CD-androstenedione complex, the formation of which was studied by differential scanning calorimetry and X-ray powder diffractometry and stoichiometry determined by 1H-nuclear magnetic resonance. A mechanism whereby CDs reduce the effective inhibitory concentrations of substrate and product as well as facilitate transport of the complexed substrate through the yeast cell wall has been suggested for the CD-promoted biotransformation.

Formation of mixed crystals in microbial conversion of sterols and steroids

Abstract The phenomenon of formation of mixed crystals during microbial conversions involving water-insoluble substrate and product was examined through the investigation of two biotransformations: (a) dehydrogenation of hydrocortisone by Arthrobacter simplex and (b) oxidation of cholesterol by Rhodococcus erythropolis . Evidence for the intrinsic ability of hydrocortisone and prednisolone molecules to form mixed crystals from both artificial solutions and microbial conversion systems was provided by x-ray powder diffractometry and differential scanning calorimetry. Mechanical agitation was shown to further promote the bioconversion of mixed crystals from an initial value of 83% prednisolone to an ultimate value of 95%. The solids in the bioconversion system of cholesterol were shown by their x-ray diffractions to be a mixture of single substrate and product crystals. A scheme of the various crystal structures likely to be formed in bioconversions—mixed single crystals, mixed crystals, and coated crystals—was presented. A solubility-based classification of the sterols and steroids with regard to their likelihood to form mixed crystals in bioconversion systems was suggested.

Phase transfer-catalyzed reduction of aromatic aldehydes by aqueous sodium formate in the presence of dichlorotris(triphenylphosphine)ruthenium(II): a kinetic study

Abstract The kinetics of transfer hydrogenation of aromatic aldehydes by aqueous sodium formate, in the presence of RuCl2(PPh3)3 and a quaternary ammonium salt, have been measured between 20 and 79 °C. Under phase transfer conditions the bicatalytic process was shown to depend on the structure and concentrations of the ammonium salt and of the aldehyde acceptor, on the concentrations of the formate donor and the metal catalyst, and to be sensitive to both the polarity of the organic solvent and the stirring rate. In a solution of concentration up to 5 M, a linear correlation was found to exist between the concentration of the aqueous formate and the reaction rate. The aldehyde substrate proved to exert an autoinhibitory effect on the catalysis. The observed activation energy, Ea = 16.7 kcal mol−1, suggests that the process is both chemically and diffusion controlled. A general reaction mechanism and rate equation have been proposed.

Microbial transformations in a cyclodextrin medium. Part 3. Cholesterol oxidation by Rhodococcus erythropolis

An intensive and systematic investigation of the oxidation of cholesterol (CL) to cholest-4-en-3-one (CN) by Rhodococcus erythropolis was undertaken in the presence of natural and chemically modified cyclodextrins (CDs) in a stirred bioreactor. The biotransformation was found to be strongly affected by the mode of addition of the natural CDs. While simultaneous addition of CL with either β- or λ-CD led to a limited enhancement effect, the microbial oxidation of β- and λ-CD complexes of CL was totally inhibited. In contrast, the alkylated CDs- dimethyl-, trimethyl- and hydroxypropyl-β-CD exhibited a remarkable enhancement of the microbial oxidation, irrespective of their mode of addition. The performance of the alkylated CDs was interpreted in the light of the measured phase solubility diagrams of CL and CN. It was thus shown that unlike the low solubilising power of hydroxypropyl-β-CD, dimethyl- and trimethyl-β-CD at 90 mm each, dissolved 9.3 and 8.7 g/l of CL and CN, respectively. Further investigation focused on the formation of CD complexes with CL and CN, analysed by X-ray powder diffractometry, differential scanning calorimetry and 1H-nuclear magnetic resonance. It was thus shown that β-CD forms a 2:1 CD:CL and CD:CN water-insoluble complexes. A mechanism of the biotransformation in homogeneous and heterogeneous CD media was presented while suggesting a direct interaction of the CD-substrate complex with microbial cells.

Transition metal-catalyzed transfer reduction of saturated aldehydes and ketones by sodium formate under phase transfer conditions

Abstract Saturated aldehydes and ketones were shown to undergo transfer hydrogenation by sodium formate under phase transfer conditions in the presence of Aliquat-336. While RuCl 2 (PPh 3 ) 3 was found to be the best catalyst for aldehyde reduction, a 1:10 mixture of RhCl(PPh 3 ) 3 and PPh 3 showed the highest activity in the transformation of ketones.

Transfer hydrogenolysis of aryl bromides by sodium formate and palladium—phosphine catalyst under phase transfer conditions

Abstract Palladium—triphenylphospine-catalyzed transfer hydrogenolysis of aryl bromides by aqueous sodium formate in a two-liquid phase system is described. Tetrahexylammonium hydrogen sulfate proved to be the best phase transfer catalyst in this system. Substitution of the H 2 O by D 2 O led to the selective transformation of the aryl bromides to monodeuterated products.

Microbial transformations in a cyclodextrin medium. Part 4. Enzyme vs microbial oxidation of cholesterol

A comparative study was conducted on two biocatalysts, resting Rhodococcus erythropolis cells and soluble cholesterol (CL) oxidase, both catalysing CL oxidation in a cyclodextrin (CD) medium. The enzyme-mediated sterol oxidation was clearly enhanced by the dimethylated β-cyclodextrin (Dimeb), as in the microbial oxidation. However, the microbial transformation was subject to a larger enhancement effect (enhancement factor of approx. 6) than the enzymic one (enhancement factor of approx. 2), with respect to the corresponding transformations with no CD. Rate vs substrate concentration curves of the microbial and enzymic systems were found to be Michaelis-Menten-like with corresponding Michaelis constant (Km) values of approx. 0.25 and 0.5 g/l. The larger Dimeb-induced effect exerted on the microbiol system was interpreted by a stronger affinity of Dimeb to the microbial cell. This CD-cell interaction was manifested through a slightly inhibited microbial growth and a limited leakage of cellular proteins and CL oxidase.

Quantitative NMR spectrometry of phase-transfer catalysts: Part 1. Determination of Extraction Constants and Water of Hydration

Abstract Proton magneticf resonance spectrometry was applied for evaluation of the distribution ratios and apparent extraction constants of tetraalkylammonium and tetraalkylphosphonium salts in two liquid-phase systems. Triethylbenzyl- and tetrabutylammonium chloride in dichloromethane and chlorobenzene, respectively, were shown to be salted out in the presence of aqueous sodium hydroxide solutions. 1 H-n.m.r. spectrometry was also used to determine the degree of hydration of quaternary onium salts in two-phase systems. The results were compatible with those obtained by the Karl Fischer method.

Bacterial toxicity of cyclodextrins: luminuous Escherichia coli as a model.

The effect of various concentrations of natural and chemically modified cyclodextrins on the luminescence of an Escherichia coli suspension was investigated. All cyclodextrins were found to reduce, albeit to a varying degree, the luminescence level of the bacterial cells, thus suggesting a direct interaction between the cyclodextrins and cells. The inhibitory concentrations IC20 and IC50 of the various cyclodextrins were determined and taken to represent their toxicity effects upon the bacterial cells. Among the natural cyclodextrins, γ- and α-CD interfered minimally with the bacterial luminescence and consequently were essentially non-toxic. The following descending order of toxicity was observed: β-CD ≫ α-CD > γ-CD. Among the chemically modified cyclodextrins, Dimeb was clearly toxic while Trimeb and the hydroxylated derivatives (hydroxypropyl-ga-CD, HPACD;-β-CD, HPBCD;-γ-CD, HPGCD) were essentially non-toxic. The following descending order of toxicity was observed: Dimeb ≫ HPBCD > Trimeb > HPACD > HPGCD.

Dehydrogenation of hydrocortisone by Arthrobacter simplex in a liposomal medium

Abstract The biotransformation of hydrocortisone to prednisolone by Arthrobacter simplex ATCC 6946 was extensively investigated in a liposomal medium, made up of multilamellar vesicles of either pure or crude egg phospholipids. The liposomal medium was found to promote faster kinetics to a higher conversion than those obtained in an aqueous medium and to be superior to water/surfactant and water/cosolvent systems, especially at high substrate loadings. The enhancing effect of the liposomal medium was partly explained by its capacity for steroid entrapment as well as by the presence of phospholipid vesicles in the bioconversion medium.