BACKGROUND Laccase mediator systems (LMS) possess great potential in organic synthesis for selective oxidation in mild conditions. However, their usage in steroid synthetic chemistry is limited by low aqueous solubility of the substrates. In this study, the effect of steroid complexation with methylated cyclodextrin (mCD) on LMS oxidation has been studied. RESULTS LMS consisted of Trametes versicolor laccase and 1‐hydroxybenzotriazole or TEMPO as redox mediators were used. The only product, ‐ 3β‐hydroxyandrost‐5‐en‐7,17‐dione (7‐keto‐DHEA) was detected during LMS oxidation of 3β‐hydroxyandrost‐5‐en‐17‐one (DHEA) at substrate concentrations within solubility. The formation of mCD complex with DHEA led to a decrease in the concentration of the reaction form of the substrate and inhibited bioconversion. When DHEA concentrations over its aqueous solubility was used, 3β,7α‐ and 3β,7β‐dihydroxyandrost‐5‐en‐17‐one (7α‐ and 7β‐OH‐DHEA) along with 7‐keto‐DHEA were formed. Both isomeric 7α‐ and 7β‐OH‐DHEA were efficiently converted to 7‐keto‐DHEA by LMS in the presence of mCD. LMS‐catalyzed oxidation of 20‐hydroxymethylpregn‐4‐en‐3‐one (HMP) in the presence of mCD resulted in a single product, pregn‐4‐en‐3‐one‐20‐carbaldehyde (20‐POA) and the reaction rate increased under elevated mCD concentrations. A preparative biochemical synthesis of 20‐POA was carried out, which has not been so far reported. CONCLUSIONS mCD affects LMS‐catalyzed oxidation of steroid alcohols. The mechanism includes formation of steroid‐mCD inclusion complexes that could inhibit, or facilitate oxidation depending on the structure of steroid‐CD complex and accessibility of the hydroxyl functions to the LMS action. © 2021 Society of Chemical Industry (SCI).
BACKGROUNDDownstream processing is an important part of industrial steroid biotechnology. The traditional recovery methods require significant amounts of the organic solvents which are associated with environmental risks. There is a need for environmentally friendly downstream processing which also may open the prospects for regeneration and repeated use of the costly materials.RESULTSMicrobial conversion of phytosterol to C17‐ketosteroids in methylated cyclodextrin (mCD) solutions was studied as a model bioprocess. Quantitative aspects of competitive complexation of mCD and natural β‐CD (β‐CD) with steroids were studied under different temperature modes and CD concentrations. The method of steroid recovery based on precipitation of insoluble β‐CD complexes ensured the complete isolation of androst‐4‐ene‐3,17‐dione, androsta‐1,4‐diene‐3,17‐dione, 9α‐hydroxyandrost‐4‐ene‐3,17‐dione, 20‐hydroxymethyl‐pregna‐1,4‐dien‐3‐one and 3β‐hydroxyandrost‐5‐en‐17‐one from aqueous media. Analysis of the 1H nuclear magnetic resonance (NMR) spectra showed a constant molar ratio of β‐CD to steroid equal to 2 in the obtained steroid β‐CD complexes. The extraction of the steroid from the β‐CD complex by small volumes of organic solvent provided a quantitative yield. The method developed ensures regeneration and repeated use of the mCDs without decrease of the basic bioconversion outputs, as was demonstrated in a series of phytosterol to androsta‐1,4‐diene‐3,17‐dione bioconversions by Mycolicibacterium neoaurum VKM Ac‐1816D.CONCLUSIONSDownstream processing based on manipulation of steroid solubility in aqueous cyclodextrin solutions is a promising alternative to traditional steroid extraction with organic solvents. This environmentally friendly approach ensures re‐use of materials without decrease in bioconversion outputs. © 2023 Society of Chemical Industry (SCI).
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