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The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ1-dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ1-dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (kcat/Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid.

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Testolactone is a potent steroid aromatase (CYP19A1) inhibitor, and its main effect is a reduction in estradiol and estrone and an increase in testosterone and androstenedione levels. In this work, we evaluated a zebrafish water tank (ZWT) as a model to investigate testolactone biotransformation and the possibility to increase knowledge regarding the applicability of the ZWT on steroid hormone elimination research, as well as on the impact of steroid hormones on the endogenous metabolism of zebrafish. High-resolution mass spectrometry combined with SIEVE software was used to discriminate the peaks of interest based on significant changes in the relative signal intensity of the m/z values between different ZWT experiments. The metabolites, 4,5-dihydrotestolactone and 1,2,4,5-tetrahydrotestolactone, the same metabolites as those described in humans, were detected in ZWT, both in quite similar proportions. The presence of testolactone in the ZWT caused a rise in testosterone and androstenedione in the water tank, similar to that in human serum. These data suggest that, while the concentration of testolactone was high enough to inhibit the aromatase enzyme, an accumulation of androgens in the water occurred, indicating that the ZWT can be considered a model to investigate the impact of steroids on live organisms.

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Using ß-sitosterol and stigmasterol as precursor materials, a concise and efficient hemisynthesis of aromatase inhibitors: testololactone and testolactone was accomplished in a well-established reaction scheme. It involves highly effective Oppaneur oxidation of both ß-sitosterol as well as stigmasterol to generate the required enone moiety in ring 'A' of the desired steroid system. The Oppaneur oxidation products of both ß-sitosterol and stigmasterol were then subjected to oxidative cleavage of the side chain to produce 4-androstene-3,17-dione. Baeyer-Villiger oxidation of 4-androstene-3,17-dione using m-CPBA yielded testololactone. Dehydrogenation of 4-androstene-3,17-dione using phenylselenyl chloride in ethyl acetate followed by selenoxide elimination with H2O2 in dichloromethane furnished androstenedienone. Baeyer-Villiger oxidation of the resulting androstenedienone yielded the desired testolactone (overall yield 33%). This expeditious reaction scheme may be exploited for the bulk production of aromatase inhibitors (especially testolactone marketed under the brand name Teslac) from the most abundant and naturally occurring phytosterols like ß-sitosterol.

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Objecave To develop a sensitive and specific LC-MS/MS method for determination of testolactone in human urine.Methods A C_(18 )column(2.1×50mm,3.5μm) was used.The mobile phase Was a mixture of acetonitrile and the buffer solution(ammonium acetate-water solution adjusted with formic acid to pH 3.5)at a flow rate of 0.5ml/min.A mass spectrometer equipped with electrospray ionization source was used as a detector and operated in the positive mode.In multiple reaction monitoring(MRM)mode,the ion transitions of m/z 301→121 and m/z 301→25 was used to qualify and quantify the testolactone,respectively.Results Chromatograms showed no endogenous interfering peaks with the urine blank sample.Each analysis was completed within 7min The calibration wag linear in the concentration range within 0.1～50μg/ml.The intra-batch and inter-batch RSD were less than 10%.The recovery rate of the extraction was about 60%.Conclusions The method is proved to meet the requirements of WADA and be suitable for routine screening.