Complete assignments of the 1H and 13C NMR spectra of testosterone and 17α‐methyltestosterone and the 1H parameters obtained from 600 MHz spectra

Hayamizu, Kikuko; Kamo, Osamu

doi:10.1002/mrc.1260280311

Cited by 30 publications

(11 citation statements)

References 26 publications

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“…The site of hydroxylation was determined by "H NMR ; the spectrum of the novel metabolite is compared with that of testosterone in Figure 4. The testosterone spectrum was assigned by two-dimensional COSY and NOESY experiments, and the results were in agreement with published assignments [46]. The "H NMR spectrum of the new metabolite produced by the CYP2D6 F%)$I mutant showed two resonances in the range 3n5-4n5 p.p.m.…”

Section: Table 2 Kinetic Parameters For the Hydroxylation Of Bufuralosupporting

confidence: 86%

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity

Smith

Modi

Pillai

et al. 1998

Biochemical Journal

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Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

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Section: Table 2 Kinetic Parameters For the Hydroxylation Of Bufuralosupporting

confidence: 86%

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity

Smith

Modi

Pillai

et al. 1998

Biochemical Journal

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“…In line with Tociu, Todasca, Bratu, Mihalache, and Manolache [17], both spectra also showed signals (two multiplets) in the range of 4.00-4.32 ppm were regarded as the methylene protons close to the COOH group as well as the signals (broad multiplets) in the range of 5.24-5.40 and 5.30-5.35 ppm which were assigned to the vinylic (�CH) protons of the double bonds of the fatty acid chains. Testosterone in the 1 H NMR spectra of HE and EE exhibited the signals at 0.69-0.99, 0.70-0.85 ppm (methyl groups), 3.72, 3.68 ppm (17α-CH close to the hydroxyl group), and 5.25-5.40, 5.30-5.37 ppm (vinylic methine protons) conforming to a previous report [18]. However, due to the overlapping peaks in 1 H NMR spectrum of WE, it is difficult to identify the components relying solely on 1 H NMR spectrum.…”

Section: Yields and Chemical Constituents Of Velvet Antlersupporting

confidence: 89%

Chemical Constituents, Antioxidant Activities, and Element Concentrations of Rusa Deer Velvet Antler Extracts

Limmatvapirat

Rodhetbhai

Somsakraksanti

et al. 2020

Journal of Chemistry

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The objective of this study was to analyze hexane extract (HE), 75% v / v ethanol extract (EE), and water extract (WE) of rusa deer velvet antlers, obtained through a three-solvent sequential extraction, for determining their chemical constituents, antioxidant activities, element concentrations, and microbial contamination. In TLC, FTIR, 1H NMR, and HPLC analysis, HE and EE showed the presence of oleic acid, linoleic acid, alpha-linolenic acid, and testosterone. EE showed the highest concentration of testosterone and the strongest antioxidant activity in DPPH and FRAP assays. In addition, the concentration of testosterone in EE was higher than in sika deer and red deer velvet antler extracts found in other studies. All extracts were composed of essential elements and had levels of toxic elements and microbial contaminants lower than the acceptable criteria set by ASEAN guidelines. Thus, EE was considered to be a safe and useful source of antioxidants and testosterone.

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“…It is probable that the binding between the steroid ring and BSA occurs at the C-3 side instead of C-17 side. In addition, based on analysis of both 1 H NMR and 13 C NMR, and comparison to the literature data (Hayamizu et al, 1990), it appears that the 17 hydroxyl group is in ␤ configuration.…”

Section: Resultsmentioning

confidence: 95%

New approach to regioselective synthesis: Using proteins in vitro as guidance—regioselective reduction of steroids with guidance from bovine serum albumin

Liu

Kayser²

2003

Biotech & Bioengineering

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A novel approach for regioselective synthesis is described. The approach uses the unique binding properties of protein as a guide to achieve regioselectivity. Specifically, steroids, e.g., 4-androstene-3,17-dione, were reduced selectively at carbon 17 using bovine serum albumin (BSA) as a regio auxiliary. The approach has resulted in a high yield (ca. 91%) in an environmental friendly aqueous medium. Both (1)H NMR and (13)C NMR data show that the BSA-assisted reduction of the steroid render the reaction regioselective compared to the control reaction. Various control reactions were performed to verify that the unique binding of BSA to steroids was the origin of regioselectivity. After one cycle of the reduction reaction, BSA was reconditioned such that it could be reused five or more times. This novel approach could be used to develop practical and "green" methods for regioselective synthesis.

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Complete assignments of the ¹H and ¹³C NMR spectra of testosterone and 17α‐methyltestosterone and the ¹H parameters obtained from 600 MHz spectra

Cited by 30 publications

References 26 publications

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity

Chemical Constituents, Antioxidant Activities, and Element Concentrations of Rusa Deer Velvet Antler Extracts

New approach to regioselective synthesis: Using proteins in vitro as guidance—regioselective reduction of steroids with guidance from bovine serum albumin

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