Cholestenone system

A 250-mI round-bottom flask fitted with a condenser (drying tube) is charged with a mixture of 2-bromocholestanone (4.7 g, 0.01 mole), lithium carbonate (7.4 g, 0.10 mole), and 100 ml of dimethylformamide. The system is flushed with nitrogen and then refluxed (mantle) for 18-24 hours. After the reflux period, the solution is cooled and poured into 500 ml of water. The aqueous mixture is extracted with 50 ml of ether, the ether extract is dried (sodium sulfate), and the ether is removed (rotary evaporator). The residue may be recrystallized from ethanol or methanol. J -Cholestenone is a white solid, mp 98-100°. 

Subsequently the work of R. F. Merritt clearly established the selectivity of additions of fluorine to double bonds in CFC13 at —78°C in preference to reactions with protons. Merritt and co-workers studied such unsaturated systems as indenes and acenapthenes (83), A4 cholestenone (84), 1,1-diphenyl-ethylene (85), Schiff s bases (86), and acetylenes (87). 

In aprotic solvents, an increase in solvent polarity resulted in an increase in the amount of ds-0-decalone formed. Similar results were also obtained in the hydrogenation of cholestenone and testosterone (see Table I). If, as suggested by McQuillin et al. (3), a more polar aprotic solvent will facilitate complexation of the carbonyl oxygen of an a,j3-unsaturated ketonic system in the same way that it increases its polarization (25), it can be assumed that what is occurring in these polar solvents is a 1,4-addition of hydrogen to the conjugated system. 

Figure 9.85 Normal phase HPLC profiles of the reaction product of the cholesterol side chain cleavage system. Peaks were identified on the basis of their retention times. (i4) Without cholesterol oxidase treatment. Cholesterol (100 nmol) was incubated with cytochrome P450scc (70 pmol) in the presence of adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Monitoring was at 214 nm. Peaks 1, cholesterol 2, pregnenolone 3, deoxycorticosterone acetate (internal standard) (B) The reaction mixture of (A) was further incubated with cholesterol oxidase at 37°C for 10 minutes. Monitoring was at 240 nm. Peaks 1, cholestenone 2, progesterone 3, deoxycorticosterone acetate (internal standard). (From Sugano et al., 1989.)...

For analytical purposes cholesterol oxidase has been immobilized on various carriers (Table 6). Electrochemical, optical, and calorimetric indication have been used as detection methods. Combination of a thermistor-coupled flow-through system with immobilized COD permitted the measurement of 0.03-0.15 mmolA cholesterol (Mattiasson et al., 1976). Ogren et al. (1980) described an immobilized COD reactor for the analysis of steroid fractions obtained by high pressure liquid chromatography. The UV absorption at 240 nm of enzymatically formed cholestenone was used as the measuring signal. Linearity was found between 10 and 80 pmol/1. 

The mitochondrial enzyme system does not exhibit a great deal of specificity, being able to oxidize cholesterol, coprostanol, cholestenone, cholestanone, coprostanone (57), and even ergosterol (58) and desmosterol (59). 

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