117407-11-1Relevant academic research and scientific papers
Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system
Agematu, Hitosi,Matsumoto, Naoki,Fujii, Yoshikazu,Kabumoto, Hiroki,Doi, Satoru,Machida, Kazuhiro,Ishikawa, Jun,Arisawa, Akira
, p. 307 - 311 (2006)
Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2α-, 2β-, 6β-, 7β-, 11β-, 12β-, 15β-, 16β-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2α-, 2β-, 6β-, 11β-, 15β-, 16α-, and 17-hydroxylation are reactions common to both human and bacterial P450s. Most of the testosterone hydroxylation catalyzed by bacterial P450s is on the β face.
A Modified Arrhenius Approach to Thermodynamically Study Regioselectivity in Cytochrome P450-Catalyzed Substrate Conversion
Luirink, Rosa A.,Verkade-Vreeker, Marlies C. A.,Commandeur, Jan N. M.,Geerke, Daan P.
, p. 1461 - 1472 (2020/03/03)
The regio- (and stereo-)selectivity and specific activity of cytochrome P450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrate relative to the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative binding free energy (ΔΔGbind) of the catalytically active substrate-binding poses, and the probability of the substrate to adopt a transition-state geometry. An established experimental method to measure activation energies of enzymatic reactions is the analysis of reaction rate constants at different temperatures and the construction of Arrhenius plots. This is a challenge for multistep P450-catalyzed processes that involve redox partners. We introduce a modified Arrhenius approach to overcome the limitations in studying P450 selectivity, which can be applied in multiproduct enzyme catalysis. Our approach gives combined information on relative activation energies, ΔΔGbind values, and collision entropies, yielding direct insight into the basis of selectivity in substrate conversion.
Preparative-Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4
Fessner, Nico D.,Srdi?, Matic,Weber, Hansj?rg,Schmid, Christian,Sch?nauer, David,Schwaneberg, Ulrich,Glieder, Anton
, p. 2725 - 2738 (2020/06/03)
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.).
Biotransformation of androst-4-ene-3,17-dione by some fungi
Yildirim, Kudret,Kuru, Ali,Keskin, Ece,Salihoglu, Aylin,Bukum, Neslihan
, p. 594 - 597 (2017/11/14)
The incubations of androst-4-ene-3,17-dione with Aspergillus candidus MRC 200634, Aspergillus tamarii MRC 72400, Aspergillus wentii MRC 200316 and Mucor hiemalis MRC 70325 for 5 days are reported. A. candidus MRC 200634 mainly hydroxylated androst-4-ene-3,17-dione at C-11α, C-15α and C-15β whilst A. wentii MRC 200316 hydroxylated it mainly at C-6β. A. tamarii MRC 72400 showed predominately a Baeyer–Villiger monooxygenase activity. M. hiemalis MRC 70325 hydroxylated the substrate at C-14α and reduced most of it at C-17.
Drug Oxidation by Cytochrome P450BM3: Metabolite Synthesis and Discovering New P450 Reaction Types
Ren, Xinkun,Yorke, Jake A.,Taylor, Emily,Zhang, Ting,Zhou, Weihong,Wong, Luet Lok
, p. 15039 - 15047 (2015/10/20)
There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.
Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution
Kille, Sabrina,Zilly, Felipe E.,Acevedo, Juan P.,Reetz, Manfred T.
scheme or table, p. 738 - 743 (2012/02/15)
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.
Structure and stereochemistry of products of hydroxylation of human steroid hormones by a housefly cytochrome P450 (CYP6A1)
Jacobsen, Neil E.,Koever, Katalin E.,Murataliev, Marat B.,Feyereisen, Rene,Walker, F. Ann
, p. 467 - 474 (2007/10/03)
The structure and stereochemistry of nine steroid metabolites isolated in quantities ranging from 0.15 to 1.8 mg were determined using a variety of NMR techniques, including heteronunclear multiple bond correlation (HMBC) using broadband adiabetic 13C pulses and phase-sensitive data presentation. Testosterone, and rostenedione and progesterone were oxidized with housefly cytochrome P450 6A1 enzyme reconstituted in vitro with housefly NADPH cytochrome P450 reductase and cytochrome b5. NMR analysis in CD3OD using a modified HMBC sequence as well as 2D heteronuclear single quantum correlation (HSQC), COSY and nuclear Overhauser and exchange spectroscopy (NOESY), combined with a detailed analysis of J couplings showed that hydroxylation occurs exclusively on the β-face of the steroids, at positions 2, 12, and 15. Copyright
Chemical synthesis of 15β-hydroxytestosterone and its derivatives using a (4-methoxyphenyl)methyl protecting group
Cerny, Ivan,Fajkos, Jan,Pouzar, Vladimir
, p. 58 - 64 (2007/10/03)
Reaction of 3β-hydroxyandrosta-5,15-dien-17-one with 4-methoxybenzyl alcohol followed by acetylation gave mainly 15β-[(4-methoxyphenyl)methoxy]- 17-oxoandrost-5-en-3β-yl acetate. This product was transformed by borohydrate reduction and organosilyl derivatization into the orthogonally protected 17β-(dimethylthexylsiloxy)-15β-[(4-methoxyphenyl)methoxy]androst- 5-en-3β-yl acetate and 17β-(dimethylisopropylsiloxy)-15β-[4- methoxyphenyl]methoxylandrost-5-en-3β-yl acetate and 17β- (dimethylisopropylsiloxy)-15β-[4-methoxyphenyl)methoxy]androst-5-en-3β-yl acetate. After deacetylation, these intermediates were submitted to Oppenauer oxidation and both yielded testosterone derivatives 17β- (dimethylthexylsiloxy)-15β-[(4-methoxyphenyl)methoxy]androst-4-en-one and 17β-(dimethylisopropylsiloxy)-15β-[(4-methoxyphenyl)-methoxy]androst-4-en- 3-one. Removal of the (4-methoxyphenyl)methyl group from position 15 by 2.3- dichloro-5,6-dicyano-1,4-benzoquinone treatment gave the partially protected derivatives 17β-(dimethylthexylsiloxy)-15β-hydroxyandrost-4-en-3-one and 17β-(dimethylisopropylsiloxy)-15β-hydroxyandrost-4-en-3-one. After acidic deprotection, the dimethylthexylsilyl derivative yielded 15β- hydroxystestosterone (15β,17β-dihydroxyandrost-4-en-3-one). Dimethylisopropylsilyl derivative was converted to the corresponding 15- hemisuccinate and 15-hemiglutarate (17β-hydroxy-3-oxoandrost-4-en-15β-yl 15-hemisuccinate and 15-hemiglutarate, respectively), which were designed as model haptens for immunoassays studies.
