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Usage

Uses

Used in Clinical and Diagnostic Testing:
6BETA-HYDROXYTESTOSTERONE is used as a certified spiking solution for various LC/MS or GC/MS applications in clinical and diagnostic testing. It serves as a starting material in calibrators or controls, aiding in the accurate measurement and analysis of testosterone and its metabolites in various samples.
Used in Endocrinology:
In the field of endocrinology, 6BETA-HYDROXYTESTOSTERONE is used as a biomarker for studying the metabolism of steroid hormones, particularly testosterone. Understanding the role and effects of this metabolite can provide insights into the hormonal balance and its impact on various physiological processes.
Used in Sports Testing:
6BETA-HYDROXYTESTOSTERONE is utilized as a reference compound in sports testing to detect the use of performance-enhancing drugs, such as testosterone. Its presence in urine samples can indicate the misuse of testosterone or other androgen steroids by athletes, helping maintain fair competition and adherence to anti-doping regulations.
Used in Pharmaceutical Research:
6BETA-HYDROXYTESTOSTERONE can be used as a starting material or reference compound in the development of new drugs targeting the androgen receptor or related metabolic pathways. Understanding its structure and properties can aid in the design of novel therapeutic agents for various medical conditions, including hormonal imbalances and androgen-related disorders.
Used in Toxicology and Drug Metabolism Studies:
6BETA-HYDROXYTESTOSTERONE is employed as a research tool in toxicology and drug metabolism studies, particularly in understanding the role of cytochrome P450 enzymes in the metabolism of endogenous and exogenous compounds. This knowledge can be applied to predict potential drug-drug interactions and optimize drug dosages for improved safety and efficacy.

InChI:InChI=1/C19H28O3/c1-18-7-5-11(20)9-15(18)16(21)10-12-13-3-4-17(22)19(13,2)8-6-14(12)18/h9,12-14,16-17,21-22H,3-8,10H2,1-2H3

Upstream product

• 63-05-8 Androstenedione 
• 58-22-0 testosterone 
• 315-37-7 testosterone heptanoate 
• 521-17-5 5-androstenediol 
• 53-43-0 dehydroepiandrosterone 
• 481-29-8 Epiandrosterone 

Downstream Products

• 899-39-8 17β-hydroxy-5α-androstane-3,6-dione 
• 2243-06-3 androst-4-ene-3,6,17-trione 

Relevant academic research and scientific papers

Preparative-Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4

Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membrane-associated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative-scale bioreactor experiment enabled the isolation of the common metabolites 6β-hydroxytestosterone and 6β-hydroxyandrostenedione on a 100 mg scale. Side-product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4-catalysed bioconversion, than the known mono-hydroxylated ones, are reported and 6-dehydro-15β-hydroxytestosterone as well as the di-hydroxylated steroids 6β,16β-dihydroxytestosterone, 6β,17β-dihydroxy-4-androstene-3,16-dione and 6β,12β-dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole-cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative-scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products. (Figure presented.).

Microbial transformation of androstenedione by Cladosporium sphaerospermum and Ulocladium chartarum

In this work, incubations of androstenedione 1 with Cladosporium sphaerospermum MRC 70266 and Ulocladium chartarum MRC 72584 have been reported. C. sphaerospermum MRC 70266 mainly hydroxylated 1 at C-6β, accompanied by a hydroxylation at C-15α, a reduction at C-17, a 5α-reduction and oxidations at C-6 and C-16 following hydroxylations. U. chartarum MRC 72584 hydroxylated 1 at C-6β, C-7α, C-7β and C-14α, accompanied by an oxidation at C-6 following its hydroxylation, a reduction at C-17 and a 5α-reduction. 6β,17β-Dihydroxyandrost-4-en-3,16-dione 8, one of the metabolites from the incubation of 1 with C. sphaerospermum MRC 70266, was determined as a new compound.

Biotransformation of testosterone by Cladosporium sphaerospermum

Incubation of testosterone 1 with Cladosporium sphaerospermum MRC 70266 afforded six metabolites and two of these metabolites, 6β,16β,17β-trihydroxyandrost-4-en-3-one 6 and 6β,12β,17β-trihydroxyandrost-4-en-3-one 7, were determined as new compounds. The fungus mainly hydroxylated testosterone 1 at C-6β, accompanied by some minor hydroxylations at C-7β, C-12β, C-15α and C-16β. A minor oxidation at C-17 and a minor 5α-reduction were also observed.

The generation of a steroid library using filamentous fungi immobilized in calcium alginate Dedicated to the memory of Professor Sir John W. Cornforth, University of Sussex (1917-2013).

Four fungi, namely, Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, and Whetzelinia sclerotiorum ATCC 18687, were subjected to entrapment in calcium alginate, and the beads derived were used in the biotransformation of the steroids 3β,17β-dihydroxyandrost-5-ene (1) and 17β-hydroxyandrost-4-en-3-one (2). Incubations performed utilized beads from two different encapsulated fungi to explore their potential for the production of metabolites other than those derived from the individual fungi. The investigation showed that steroids from both single and crossover transformations were typically produced, some of which were hitherto unreported. The results indicated that this general technique can be exploited for the production of small libraries of compounds.

Microbial transformation of epiandrosterone by Aspergillus sydowii

Incubation of epiandrosterone with Aspergillus sydowii MRC 200653 afforded ten metabolites. The fungal dehydrogenation of epiandrosterone is reported for the first time. The formation of the major metabolite, 6?-hydroxyandrost-4-ene-3,17-dione, involved first dehydrogenation to give a 4-ene and then hydroxylation at C-6?. Small amounts of the substrate were hydroxylated at C-1α, C-7α, C-7β and C-11α.

Biotransformation of testosterone and testosterone heptanoate by four filamentous fungi

The microbial transformations of testosterone and testosterone heptanoate by four fungi: Absidia griseolla var. igachii PTCC 5260, Acremonium chrysogenu PTCC 5271, Fusarium fujikuroi PTCC 5144, and Fusarium solani complex PTCC 5285 were investigated for the first time. Incubation of testosterone heptanoate with F. fujikuroi and F. solani yielded three metabolites, which were isolated and characterized as testosterone, androst-4-ene-3,17-dione, and 6β-hydroxy testosterone. 6β-Hydroxy testosterone was the major metabolite obtained from testosterone heptanoate biotransformation by two fungal species. A. griseolla and A. chrysogenu produced 14α-hydroxy testosterone as major metabolite, together with testosterone and 6β-hydroxy testosterone in lower yields. The biotransformation of testosterone by F. fujikuroi and A. griseolla was also investigated in order to examine the influence of the ester group on the course of transformation. Androst-4-ene-3,17-dione was only identified in the biotransformation of testosterone by F. fujikuroi. The same product was observed in incubation of testosterone by A. griseolla, together with 14α-hydroxy testosterone in very low yield. Furthermore, time course study was also carried out in order to examine the formation of metabolites as a function of time, which was determined by HPLC. The structures of compounds were determined by their comprehensive spectroscopic analysis and comparison with literature data.

Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation

The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone - just as other steroids - is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β-position, while the slow formation of 6β-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism. 400 cytochrome P450 2D6 (CYP2D6) variants representing all possible amino acid exchanges at two important enzyme's residues were expressed and individually analyzed to investigate their influence on regioselective steroid hydroxylation. Steroids represent a substrate class atypical for wildtype CYP2D6. Employing this strategy CYP2D6 variants with improved activity and variants with altered region-preference were identified and characterized.

Cytochrome P450 metabolic activities in the small intestine of cynomolgus macaques bred in cambodia, china, and indonesia

Summary: Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.

Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C - H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C - H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96 - 97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product-6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.