HYDROPHOBIC AROMATIC THIO (DITHIO)COMPOUNDS AS CHROMOGENIC ANALYTICAL REAGENTS FOR PHOTOMETRIC DETERMINATION OF THIOLS BY MERCAPTO GROUP II. A UNIFIED MOLECULAR MODEL OF THE ANALYTICAL REAGENT

The concept of a unified molecular model is proposed for the directed synthesis of chromogenic analytical reagents for the mercapto group in ("thiol-thiol") substitution reactions (SH reagents). The unified molecular model of the SH reagent includes an aromatic chromophore-containing thio fragment covalently bound by an ordinary bond on a sulfide sulfur atom to an electrophilic, relatively soft one [in terms of the principle of hard and soft acids and bases (the HSAB principle)] reaction center. The characteristics of the analytical effect of the SH reagent are determined by the structure of the thio fragment. The required change in the reactivity and selectivity of the SH reagent are achieved mainly by varying the softness (stiffness) of the reaction center. Spectral characteristics of analytical effects, reactivity and selectivity of SH reagents (obtained earlier and synthesized for the first time in this work) – hydrophobic aromatic thio (dithio) compounds: thiocyanates, symmetric disulfides, and thiomercuric compounds – were studied in an aprotic dipolar DMF medium. In support of the concept of the unified molecular model for compounds containing identical thiofragments it was established: - spectral characteristics of the analytical effect of all three types of compounds (thiocyanates, disulfides and thiomercuric compounds) are the same in the DMF medium. - in accordance with the principle of HSAB, the reactivity increases in the sequence thiocyanate < disulfide <<thiomercuric compound, which corresponds to an increase in the softness of the reaction center in the series: -С << - S-<< -Hg-. The universality of the concept of the unified molecular model follows from consideration of the spectral and kinetic characteristics not only of Ellman reagent, but as well of thio(dithio)azo compounds, thiocyanate and thiomercuric compound containing the same 3-carboxy, 4-nitrophenylthio fragment.

3 α-Hydroxysteroid-5β-oxidoreductase in Tissue Cultures of Digitalis lanata

Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5β-pregnane-3,20-dione or 5β-pregnan-3β-ol-20-one, were administered to light- or darkgrown shoot cultures of Digitalis lanata. The unsaturated com pounds were reduced to their respective 5 a-pregnanes, 5β-pregnane-3,20-dione was reduced to 5β-pregnan-3α-ol-20-one and 5β-pregnan-3β-ol-20-one was isomerized to the respective 3α-pregnane. Suspension cultures of Digitalis lanata, on the other hand, accumulated both the 3α- and the 3β-isom er of 5β-pregnan-3-ol-20-one when incubated in the presence of 5β-pregnane- 3.20-dione. When 5β-pregnan-3α-ol-20-one was administered the cultured cells accumulated large amounts of the 3β-isomer together with small amounts of 5β-pregnane-3,20-dione, which may be regarded as an intermediate during the isomerization reaction. Cell-free, buffered extracts from light-grown shoots were shown to reduce 5β-pregnane- 3.20-dione almost exclusively to 5β-pregnan-3α-ol-20-one when 0.05 m MgCl2 were present in the incubation mixture. Under these conditions the formation of 5β-pregnan-3β-ol-20-one was inhibited. The enzyme activity could be recovered from m em brane-free supernatants. Optimum enzyme activity occurred at pH 7.0 and 42 °C. The energy of activation was 56.2 kJ/mol and the enzyme reaction was found to be NADPH -dependent. SH reagents were essential for enzyme activity. The enzyme seems to be specific for 5β-pregnan-3-ones since neither 5 a-pregnane-3-ones nor Δ4/Δ5-pregnenes were reduced. The NADPH : 5β-pregnane 3α-hydroxysteroid-5β-oxidoreductase described here may play a role in the regulation of cardenolide biosynthesis by removing precursors, such as 5β-pregnane-3,20-dione, from the pathway