Steroid sulfatase inhibitor alters blood pressure and steroid profiles in hypertensive rats

Our hypothesis is that the steroid sulfatase gene (Sts) may indirectly contribute to the modulation of blood pressure (BP) in rats with genetic hypertension. The steroid sulfatase enzyme (STS) catalyzes the conversion of estrone sulfate, dehydroepiandrosterone sulfate, cholesterol sulfate and glucocorticoid sulfates to their active nonconjugated forms. This causes the elevation of biologically active steroids, such as glucocorticoids, mineralcorticoids as well as testosterone, which may lead to increased BP. The main objective was to examine the effects of a steroid sulfatase inhibitor on blood pressure and steroid levels in rats with hypertensive genetic backgrounds. Three treatment groups, 5-15 weeks of age were used: controls, estrone and STS inhibitor (estrone-3-O-sulfamate), (n=8 per group). BP was taken weekly by tail cuff, and serum testosterone (T), estrogens (E), and plasma corticosterone (C) levels were measured by radioimmunoassay. BP was significantly reduced by the STS inhibitor in the strains with genetically elevated BP. Also the inhibitor alone significantly reduced plasma corticosterone in all strains compared to estrone treatment with a concomitant as well as significant rise in estrogens and reduction in testosterone and body weight.     

The difluoromethylene group as a replacement for the labile oxygen in steroid sulfates: a new approach to steroid sulfatase inhibitors

Several estrone sulfate and estradiol sulfate analogues, in which the sulfate group was replaced with an alpha,alpha-difluoromethylenesulfonate group or an alpha,alpha-difluoromethylenetetrazole group, were examined as inhibitors of steroid sulfatase (STS). These compounds were 4.5-10.5 times more potent than their non-fluorinated analogues. Moreover, the presence of the fluorines changed the mode of inhibition from mixed to competitive. The inhibitor bearing the alpha,alpha-difluoromethylenetetrazole group exhibited an affinity for STS approaching that of the natural STS substrate, estrone sulfate. Possible reasons for the enhanced affinity of the fluorinated compounds compared to their non-fluorinated counterparts are discussed.     

Steroid sulfatase activity in the rat ovary, cultured granulosa cells, and a granulosa cell line

Direct production of gonadal steroids from sulfated adrenal androgens may be an important alternative or complementary pathway for ovarian steroidogenesis. The conversion of sulfated adrenal androgens, present in serum at micromolar concentrations in adult women, into unconjugated androgens or estrogens requires steroid sulfatase (STS) activity. STS activity has not been characterized in the rat ovary. Substantial STS activity was present in homogenates of rat ovaries, primary cultures of rat granulosa cells, and a granulosa cell line, as determined by conversion of radiolabeled estrone sulfate (E₁S) to unconjugated estrone. The potent inhibitor estrone sulfamate eliminated the STS activity. Using E₁S as a substrate with microsomes prepared from a granulosa cell line, the K_m of STS activity was approximately 72 μM, a value in agreement with previously published data for rat STS. Therefore, ovarian cells possess STS and can remove the sulfate from adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S). Using DHEA-S as a steroidogenic substrate represents an alternative model for the production of ovarian steroids versus the “two cell, two gonadotropin” model of ovarian estrogen synthesis, whereby thecal cells produce androgens from substrate cholesterol and granulosa cells convert the androgens into estrogens. The relative contribution of STS activity to ovarian steroidogenesis remains unclear but may have important physiological and pathophysiological implications.     

Development of novel steroid sulfatase inhibitors; II. TZS-8478 potently inhibits the growth of breast tumors in postmenopausal breast cancer model rats

In postmenopausal breast cancer tissue, steroid sulfatase (STS) activity is high and much estrone sulfate also exists; these facts reveal that estrone sulfate may be involved in the growth of breast cancer as an estrogen source. Steroid sulfatase is an enzyme, which catalyzes hydrolysis from estrone sulfate to estrone, and the development of steroid sulfatase inhibitors is expected as novel therapeutic drugs for postmenopausal breast cancer. We have developed a novel compound 2',4'-dicyanobiphenyl-4-O-sulfamate (TZS-8478), which has potent steroid sulfatase-inhibitory activity and exhibits no estrogenicity in vitro and in vivo. To elucidate its usefulness as a therapeutic drug for postmenopausal breast cancer, we examined the breast cancer cell proliferation- and breast tumor growth-inhibitory activity of TZS-8478 in postmenopausal breast cancer model rats. TZS-8478 dose-dependently suppressed the estrone sulfate-stimulated proliferation of MCF-7 cells. Regarding nitrosomethylurea (NMU)-induced postmenopausal breast cancer models, furthermore, TZS-8478 (0.5 mg/kg per day) markedly inhibited the estrone sulfate-stimulated growth of breast tumors similarly to estrone sulfate-depletion. TZS-8478 completely inhibited steroid sulfatase activity in tumor, uterus and liver, and also markedly lowered plasma concentrations of estrone and estradiol. The above mentioned results suggested that TZS-8478 may be useful as a therapeutic drug for estrogen-dependent postmenopausal breast cancer.     

Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.     

D-ring modified estrone derivatives as novel potent inhibitors of steroid sulfatase

A series of novel D-ring modified derivatives of estrone was synthesized and tested as inhibitors of steroid sulfatase (STS). The steroidal D-ring was cleaved via an iodoform reaction and thermal condensation of the resulting marrianolic acid derivative gave 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives, where a piperidinedione moiety is in place of the D-ring. This synthetic approach was found to give a higher overall yield than the literature method of Beckmann rearrangement. A range of alkyl side chains have been introduced on the nitrogen atom of the imido-ring and the corresponding 3-O-sulfamates synthesized. The new D-ring modified estrone derivatives bearing a propyl (39) and a 1-pyridin-3-ylmethyl (46) moiety had IC(50) values of 1 nM when tested in placental microsomes for the inhibition of STS. These compounds are therefore up to 18-fold more potent than EMATE, the very first highly potent irreversible steroidal STS inhibitor.     

Multipoint linkage analysis of steroid sulfatase (X-linked ichthyosis) and distal Xp markers

Six families with steroid sulfatase deficiency (STS; X-linked ichthyosis) have been studied with the Xg blood group (XG) and the DNA markers dic56 (DXS143), 782 (DXS85), pD2 (DXS43), and GMGX9. Carrier status of females was determined by assay of STS in hair roots. GMGX9 detects a frequent restriction fragment length polymorphism and also identifies a deletion in the majority of families with STS deficiency, including five of the six reported here. The linkage relationship of this marker to the others was studied in normal three-generation families yielding 32 phase-known meioses informative for two or more markers. No recombinants were observed between STS and GMGX9, giving a maximum lod score of 8.73 at zero recombination. Multipoint linkage analysis taking STS and GMGX9 as a single locus and incorporating two-point marker data and STS-XG data from published studies gave the map (Sequence: see text). This order was 2.4 times more likely than with (STS,GMGX9) and dic56 reversed and is supported by our findings in a male with steroid sulfatase deficiency due to a deletion of Xp22.3 which encompasses the XG locus. He is deleted for GMGX9 but shows normal hybridization to dic56 and 782.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.     

4,4'-Benzophenone-O,O'-disulfamate: a potent inhibitor of steroid sulfatase

We investigated whether the benzophenone moiety can be used as core element of steroid sulfatase (STS) inhibitors. While 4- and 3-benzophenone-O-sulfamates inhibit STS with IC(50) values between 5 and 7 microM irrespective of additional hydroxy and methoxy substituents at the second phenyl ring, benzophenone-O,O'-disulfamates show increased activity. With an IC(50) value of 190 nM the 4,4'-derivative is the first small monocyclic STS inhibitor coming close to the potency of the steroidal standard estrone sulfamate.     

Inactivation of recombinant human steroid sulfatase by KW-2581

Steroid sulfatase (STS) catalyses the hydrolysis of the sulfate esters of 3-hydroxy steroids, which are inactive transport or precursor forms of the active 3-hydroxy steroids. STS inhibitors are expected to block the local production and, consequently to reduce the active steroid levels; therefore, they are considered as potential new therapeutic agents for the treatment of estrogen- and androgen-dependent disorders such as breast and prostate cancers. KW-2581 is a novel steroidal STS inhibitor. In the present study, we found KW-2581 inhibited recombinant human STS (rhSTS) activity with an IC(50) of 2.9 nM when estrone sulfate was used as a substrate. The potency of KW-2581 was approximately 5-fold higher than that of a non-steroidal STS inhibitor, 667 COUMATE. KW-2581 was able to equally inhibit rhSTS activity when dehydroepiandrosterone sulfate was used as another substrate. KW-2581 inhibited rhSTS activity in a time- and concentration-dependent manner (k(inact), 0.439 min(-1); K(i, app), 15 nM), suggesting that it is an active site-directed irreversible inhibitor. Both decrease of KW-2581 concentration and increase of the des-sulfamoylated form's concentration were simultaneously observed during the reaction in a time-dependent manner with corresponding to the decrease of STS activity. Our findings for the first time demonstrated the production of des-sulfamoylated form of the compound as a consequence of STS inactivation.     

Inhibition of steroid sulfatase with 4-substituted estrone and estradiol derivatives

Steroid sulfatase (STS) catalyzes the desulfation of biologically inactive sulfated steroids to yield biologically active desulfated steroids and is currently being examined as a target for therapeutic intervention for the treatment of breast cancer. We previously demonstrated that 4-formyl estrone is a time- and concentration-dependent inhibitor of STS. We have prepared a series of 4-formylated estrogens and examined them as irreversible STS inhibitors. Introducing a formyl, bromo or nitro group at the 2-position of 4-formylestrone resulted in loss of concentration and time-dependent inhibition and a considerable decrease in binding affinity. An estradiol derivative bearing a formyl group at the 4-position and a benzyl group at the 17β-position yielded a potent concentration and time-dependent STS inhibitor with a K(I) of 85 nM and a k(inact) of 0.021 min(-1) (k(inact)/K(I) of 2.3 × 10(5)M(-1)min(-1)). Studies with estrone or estradiol substituted at the 4-position with groups other than a formyl group revealed that good reversible inhibitors can be obtained by introducing small electron withdrawing groups at this position. An estradiol derivative with fluorine at the 4-position and a benzyl group at the 17β-position yielded a potent, reversible inhibitor of STS with an IC(50) of 40 nM. The introduction of relatively small electron withdrawing groups at the 4-position of estrogens and their derivatives may prove to be a general approach to enhancing the potency of estrogen-derived STS inhibitors.     

Estrone 3-sulfate mimics,inhibitors of estrone sulfatase activity: homology model construction and docking studies

Steroid sulfatase (STS) is a new target for the endocrine therapy of breast cancer. To ascertain some of the requirements for inhibition of estrone sulfatase activity, a number of novel analogues of estrone 3-O-sulfate possessing sulfate surrogates were synthesized and evaluated as inhibitors of estrone sulfatase (STS) in comparison to a lead inhibitor, estrone-3-O-methylthiophosphonate (E1-3-MTP). Using a selective enzyme digestion, one of the diastereoisomers of this compound, (R(p))-E1-3-MTP, could be prepared and evaluated. From structure-activity studies, we show that chirality at the phosphorus atom, hydrophobicity, basicity, size, and charge all influence the ability of a compound to inhibit estrone sulfatase activity. Of these, hydrophobicity seems to be the most important since simple, active nonsteroidal inhibitors, based on 5,6,7,8-tetrahydronaphth-2-ol (THN), can be prepared, provided that they are lipophilic enough to partition into a nonpolar environment. Also, a negatively charged group is favorable for optimal binding, although it appears that the presence of a potentially cleavable group can compensate for lack of charge in certain cases. A homology model of STS has been constructed from the STS sequence, and molecular docking studies of inhibitors have been performed to broaden the understanding of enzyme/inhibitor interactions. This model clearly shows the positions of the key amino acid residues His136, His290, Lys134, and Lys368 in the putative catalytic region of the formylglycine at position 75, with residues Asp35, Asp36, Asp342, and Gln343 as ligands in the coordination sphere of the magnesium ion. Docking studies using the substrate and estrone-3-sulfate mimics that are active inhibitors indicate they are positioned in the area of proposed catalysis, confirming the predictive power of the model.     

Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC₅₀ below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC₅₀ below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 μM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.     

Steroid sulfatase inhibitors: promising new tools for breast cancer therapy?

Inhibition of aromatase is currently well-established as the major treatment option of hormone-dependent breast cancer in postmenopausal women. However, despite the effects of aromatase inhibitors in both early and metastatic breast cancer, endocrine resistance may cause relapses of the disease and progression of metastasis. Thus, driven by the success of manipulating the steroidogenic enzyme aromatase, several alternative enzymes involved in steroid synthesis and metabolism have recently been investigated as possible drug targets. One of the most promising targets is the steroid sulfatase (STS) which converts steroid sulfates like estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) to estrone (E1) and dehydroepiandrosterone (DHEA), respectively. Estrone and DHEA may thereafter be used for the synthesis of more potent estrogens and androgens that may eventually fuel hormone-sensitive breast cancer cells. The present review summarizes the biology behind steroid sulfatase and its inhibition, the currently available information derived from basic and early clinical trials in breast cancer patients, as well as ongoing research. Article from the Special Issue on Targeted Inhibitors.     

Anti-cancer activities of novel D-ring modified 2-substituted estrogen-3-O-sulfamates

Sulfamoylated derivatives of the endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2 (7)), such as 2-methoxy-3-O-sulfamoyl estrone (2-MeOEMATE (1)), display greatly enhanced activity against the proliferation of human cancer cells and inhibit steroid sulphatase (STS), another current oncology target. We explore here the effects of steroidal D-ring modification on the activity of such 2-substituted estrogen-3-O-sulfamates in respect of inhibition of tumour cell proliferation and steroid sulphatase. The novel 17-deoxy analogues of 2-MeOEMATE and the related 2-ethyl and 2-methylsulfanyl compounds showed greatly reduced inhibition of MCF-7 proliferation. Introduction of a 17alpha-benzyl substituent to such 2-substituted estrogen sulfamates also proved deleterious to anti-proliferative activity but could, in one case, enhance STS inhibition with respect to the parent substituted estrone sulfamate. In contrast, selected 17-oxime derivatives of 2-MeOEMATE displayed an enhanced anti-proliferative activity. These results illustrate that enhanced in vitro anti-cancer activity can be achieved in the 2-substituted estrogen sulfamate series and highlight, in particular, the importance of potential hydrogen bonding effects around the steroidal D-ring in the activity of these molecules. The SAR parameters established herein will assist the future design of anti-proliferative and anti-endocrine agents as potential therapeutics for both hormone dependent and independent cancers.     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Boronic acids as inhibitors of steroid sulfatase

Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment of steroid-dependent cancers. Two steroidal and two coumarin- and chromenone-based boronic acids were synthesized and examined as inhibitors of purified STS. The boronic acid analog of estrone sulfate bearing a boronic acid moiety at the 3-position in place of the sulfate group was a good competitive STS inhibitor with a K_i of 2.8 μM at pH 7.0 and 6.8 μM at pH 8.8. The inhibition was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. An estradiol derivative bearing a boronic acid group at the 3-position and a benzyl group at the 17-position was a potent reversible, non-competitive STS inhibitor with a K_i of 250 nM. However, its 3-OH analog, a known STS inhibitor, exhibited an almost identical affinity for STS and also bound in a non-competitive manner. It is suggested that these compounds prefer to bind in a hydrophobic tunnel close to the entrance to the active site. The coumarin and chromenone boronic acids were modest inhibitors of STS with IC₅₀s of 86 and 171 μM, respectively. Surprisingly, replacing the boronic acid group of the chromenone derivative with an OH group yielded a good reversible, mixed type inhibitor with a K_i of 4.6 μM. Overall, these results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog.     

Novel D-ring modified steroid derivatives as potent,non-estrogenic,steroid sulfatase inhibitors with in vivo activity

In pursuit of novel steroid sulfatase (STS) inhibitors devoid of estrogenicity, several D-ring modified steroid derivatives were synthesised. In vitro evaluation of the compounds identified two highly potent inhibitors, 4a and 4b, which were 18 times more active than estrone-3-O-sulfamate (EMATE), both having IC(50) values of ca. 1nM. These 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives were synthesised from estrone, via the intermediate 1, which was easily alkylated, deprotected and sulfamoylated affording the final compounds in high yields. In order to assess their biological profile, the selected inhibitors were tested for their in vivo inhibitory potency and estrogenicity in ovariectomised rats. After an oral dose of 10mg/kg per day for 5 days, 4a and 4b were found to inhibit rat liver steroid sulfatase by 99%. They were also devoid of estrogenic activity in the uterine weight gain assay, indicating that these two leads have therapeutic potential for the treatment of hormone-dependent breast cancer.     

Steroid sulfatase and estrogen sulfotransferase in normal human tissue and breast carcinoma

Steroid sulfatase (STS) hydrolyzes inactive estrone sulfate (E1-S) to estrone (E1), while estrogen sulfotransferase (EST; SULT 1E1 or STE gene) sulfonates estrogens to estrogen sulfates. They are considered to play important roles in the regulation of local estrogenic actions in various human tissues, however, their biological significance remains largely unknown. Therefore, we examined the expression of STS and EST in non-pathologic human tissues and breast carcinomas. STS expression was very weak except for the placenta, while EST expression was markedly detected in various tissues examined. In breast carcinoma tissues, STS and EST immunoreactivity was detected in carcinoma cells in 74 and 44% of cases, respectively, and was significantly associated with their mRNA levels and enzymatic activities. STS immunoreactivity was significantly correlated with the tumor size, and an increased risk of recurrence. EST immunoreactivity was inversely correlated with the tumor size or lymph node status. Moreover, EST immunoreactivity was significantly associated with a decreased risk of recurrence or improved prognosis. Our results suggest that EST is involved in protecting various peripheral tissues from excessive estrogenic effects. In the breast carcinoma, STS and EST are suggested to play important roles in the regulation of in situ estrogen production in the breast carcinomas.     

Enzymatic reduction of dehydrocholic acid to 12-ketochenodeoxycholic acid with NADH regeneration

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from dehydrocholic acid. The specific reduction of dehydrocholic with NADH was catalysed by 3α-hydroxysteroid dehydrogenase (3α-hydroxysteroid: NAD(P)⁺ oxidoreductase, EC 1.1.1.50) and 7α-hydroxysteroid dehydrogenase (7α-hydroxysteroid:NAD⁺ 7-oxidoreductase, EC 1.1.1.159). Cofactor regeneration was obtained through the formate dehydrogenase (formate:NAD⁺ oxidoreductase, EC 1.2.1.2) catalysed oxidation of formate. Complete transformation of dehydrocholic acid to the 12-keto derivative was achieved with a coenzyme turnover number up to 1200. No steroid by-products were detected by high performance liquid chromatography and thin layer chromatography. The process yielded 9 g product l^?1 in 66–84 h. The high purity of the enzymatically prepared 12-ketochenodeoxycholic acid should drastically reduce the formation of the toxic by-product lithocholic acid, which occurs in the synthesis of chenodeoxycholic acid when using chemical methods alone.