The action pattern of Penicillium lilacinum dextranase.

The product distributions resulting from the action of Penicillium lilacinum dextranase on end-labelled oligosaccharides of the isomaltose series have been determined. The initial rates of formation of labelled products were measured for isomaltotriose up to isomalto-octaose, and the molar proportions and radioactivity of the final products from isomaltotriose up to isomaltohexaose were determined. D-Glucose was released only from isomaltotriose and isomaltotetraose, by hydrolysis of the first linkage from the reducing end (linkage 1); the terminal bonds of higher members of the series were not attacked. All oligosaccharides except isomaltotriose were hydrolyzed at more than one linkage. The main points of attack on isomaltotetraose up to isomalto-octaose were at linkage 2, and at the third linkage from the non-reducing end; these two positions coincide for isomaltopentaose. The degradation of isomaltotriose up to isomalto-octaose was entirely hydrolytic. The enzyme also catalyzed an extremely slow, concentration-dependent degradation of isomaltose, and this may have occurred via a condensation to isomaltotetraose, followed by hydrolysis of linkage 1 to give D-glucose and isomaltotriose.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

Expedited Baeyer-Villiger oxidation of steroidal ketones by microwave irradiation

Microwave (MW) assisted reactions are currently having considerable importance in the synthesis of organic compounds. Considering the remarkable application of Baeyer–Villiger (BV) reaction in the synthesis of natural products and steroid–peptide conjugates, we report here some of our findings of BV oxidation of carbonyl compounds with special reference to steroidal ketones under MW irradiation justifying its accelerating effect.     

Baeyer-Villiger oxidation of bicyclic ketones by Cylindrocarpon destructans ATCC 11011

Baeyer-Villiger oxidation of bicyclic ketones1a,1b and4 using whole cell suspensions of the fungusCylindrocarpon destructans was found to proceed quantitatively and in case of substrate (±)-1b a moderate enantioselectivity was observed leading to (1S,6R)-1b and (1R,6S)-2b with 28% and 27% e.e., respectively.     

Gas chromatographic determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue.

A gas chromatographic method has been presented for the determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue. The procedure utilized radioactive tracers added to homogenate for correcting methodological loss, preliminary separation of steroids by thinlayer chromatography, acetylation, rechromatography on chromatoplate and gas chromatography on 3% SE-30 or 1% XE-60 columns with flame ionisation detection of steroids by using internal standards. Results of control experiments and representative clinical findings on normal and polycystic ovaries are reported.     

Biotransformation of cycloalkenones by fungi Baeyer-Villiger oxidation of bicycloheptenone by dematiaceous fungi

Summary The biotransformation by ring expansion of a bicyclo [3.2.0]-alkenone has been demonstrated in 29 species of dematiaceous fungi. One of these biocatalysts,Curvularia lunata NRRL 2380 has been shown to produce synthetically-important chiral lactones in high yields.     

Biotransformation of dehydroepiandrosterone (DHEA) with Penicillium griseopurpureum Smith and Penicillium glabrum (Wehmer) Westling

Microbial transformation of dehydroepiandrosterone (DHEA, 1) using Penicillium griseopurpureum Smith and Penicillium glabrum (Wehmer) Westling has been investigated. Neither fungi had been examined previously for steroid biotransformation. One novel metabolic product of DHEA (1) transformed with P. griseopurpureum Smith, 15α-hydroxy-17a-oxa-d-homo-androst-4-ene-3,17-dione (5), was reported for the first time. The steroid products were assigned by interpretation of their spectral data such as ¹H NMR, ¹³C NMR, IR, and HR-MS spectroscopy. P. griseopurpureum Smith was proven to be remarkably efficient in oxidation of the DHEA (1) into androst-4-en-3,17-dione (2). The strain was also observed to yield different monooxygenases to introduce hydroxyl groups at C-7α, -14α, and -15α positions of steroids. Preference for Baeyer–Villiger oxidation to lactonize D ring and oxidation of the 3β-alcohol to the 3-ketone were observed in both incubations. The strain of P. glabrum (Wehmer) Westling catalyzed the steroid 1 to generate both testololactone 3, and d-lactone product with 3β-hydroxy-5-en moiety 8. In addition, the strain promoted hydrogenation of the C-5 and C-6 positions, leading to the formation of 3β-hydroxy-17a-oxa-d-homo-5α-androstan-3,17-dione (9).The biotransformation pathways of DHEA (1) with P. glabrum (Wehmer) Westling and P. griseopurpureum Smith have been investigated, respectively. Possible metabolic pathways of DHEA (1) were proposed.     

First enantiodivergent Baeyer-Villiger oxidation by recombinant whole-cells expressing two monooxygenases from Brevibacterium

Microbial Baeyer-Villiger oxidations of representative mesomeric ketones with recombinant Escherichia coli cells expressing two monooxygenases from Brevibacterium were investigated. The two enzymes displayed enantiodivergent biotransformations on an array of structurally diverse substrates, allowing access to some key lactone intermediates in natural compound synthesis.     

Conversion of pregnenolone to DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17alpha-hydroxypregnenolone.

Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17alpha-OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK-293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17alpha-OHPreg, and that, subsequently, the accumulated 17alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17alpha-OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17alpha-OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA.     

An efficient enzymatic Baeyer-Villiger oxidation by engineered Escherichia coli cells under non-growing conditions

Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.