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| Targets |
16-Dehydroprogesterone serves as a substrate for 16-dehydroprogesterone reductase (also referred to as Δ16-steroid reductase) in Eubacterium sp. strain 144. No IC₅₀, Ki, EC₅₀, or DC₅₀ values are reported in the literature. [1]
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| ln Vitro |
- In cell suspensions of Eubacterium sp. strain 144, 16-Dehydroprogesterone is reduced to 17-isoprogesterone. The reaction rate is initially very low under standard conditions (pH 7.0, 1% methanol). Optimization of pH to 5.5 (potassium phosphate buffer) and increasing methanol concentration to 10% (vol/vol) does not significantly enhance the activity. [1]
- Growth of strain 144 in the presence of hemin (5 μg/ml) stimulates 16-Dehydroprogesterone reductase activity approximately fourfold during the first 30 minutes of incubation, resulting in a biphasic time course. The biphasic kinetics are eliminated when an exogenous electron donor (H₂ or pyruvate at 10 mM) is provided, leading to near-complete conversion of 16-Dehydroprogesterone to 17-isoprogesterone within 20–30 minutes. Without electron donors, activity remains low (e.g., 15 nmol 17-isoprogesterone/mg protein in 30 min). With H₂, activity reaches 143 nmol/mg protein; with pyruvate, 111 nmol/mg protein. [1] - In the presence of H₂ or pyruvate, 17-isoprogesterone formed from 16-Dehydroprogesterone is further transformed to products lacking UV absorbance at 254 nm, indicating reduction of the C4-C5 double bond (ring A) by progesterone reductase. [1] - When 16-Dehydroprogesterone (256 μM) is incubated with cell suspensions of strain 144 grown with hemin and 16-Dehydroprogesterone under H₂, the steroid is rapidly hydrated to 16α-hydroxyprogesterone (detectable at <30 s), then reduced to 17-isoprogesterone; after 30 min, both 16-Dehydroprogesterone and 16α-hydroxyprogesterone are depleted, and 17-isoprogesterone is further metabolized. [1] |
| ln Vivo |
No direct in vivo studies on 16-Dehydroprogesterone are described in the provided literature. The reference mentions that Eubacterium sp. strain 144 converts 16α-hydroxyprogesterone to 17-isoprogesterone in rat intestinal tracts, with 16-Dehydroprogesterone as an intermediate, but no in vivo administration or activity data for 16-Dehydroprogesterone itself are presented. [1]
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| Enzyme Assay |
- 16-Dehydroprogesterone reductase assay in cell suspensions: Cells of Eubacterium sp. strain 144 were harvested (centrifugation at 5,500 × g for 15 min at 0°C) and resuspended in anaerobic 50 mM potassium phosphate buffer (pH 5.5) containing 1 mM dithiothreitol to achieve a cell protein concentration of approximately 1.26–1.62 mg/ml. The standard assay mixture (4.0 ml) contained 0.8–1.3 mg cell protein per ml, 50 mM potassium phosphate buffer (pH 5.5), 1 mM dithiothreitol, 10% (vol/vol) methanol, and 256 μM 16-Dehydroprogesterone. The reaction was carried out at 37°C under anaerobic conditions (O₂-free Ar or H₂). For H₂ or pyruvate stimulation, cells were preincubated with the electron donor (10 mM pyruvate or H₂ atmosphere) for 30 min before adding the steroid. At intervals, 0.5 ml samples were withdrawn, mixed with 0.5 ml of 0.5 N H₃PO₄ to stop the reaction, and steroids were extracted with diethyl ether. Products were separated by high-pressure liquid chromatography and quantified by UV absorbance at 254 nm. [1]
- Hydrogenase activity confirmation: Hydrogenase activity in strain 144 was confirmed by observing H₂-dependent reduction of methylene blue (no activity under Ar). Similarly, pyruvate metabolism was indicated by methyl viologen reduction only in the presence of pyruvate. [1] |
| Cell Assay |
- Bacterial growth and cell suspension preparation: Eubacterium sp. strain 144 was grown in a medium containing tryptic soy broth without glucose, yeast extract, and L-arginine, with Na₂S as reducing agent (cysteine omitted). The medium was modified by adding 16-Dehydroprogesterone (32 μM final concentration, from a stock solution in methanol) and/or hemin (5 μg/ml). Cultures were incubated at 37°C under 100% CO₂. Cells were harvested during early log phase (absorbance at 660 nm = 0.15–0.25, corresponding to 17–18 h incubation). The cell pellet was suspended in anaerobic 50 mM potassium phosphate buffer (pH 5.5) containing 1 mM dithiothreitol. The suspension was diluted 1:10 to give a turbidity of 85–100 Klett units (red filter), equivalent to about 1.26 mg cell protein/ml (without hemin) or 1.62 mg/ml (with hemin). All procedures were performed under O₂-free Ar. [1]
- Effect of hemin and electron donors on steroid reductase activity: Cells grown without hemin or without 16-Dehydroprogesterone showed minimal or no detectable 16-Dehydroprogesterone reductase or progesterone reductase activities. Maximum activities required growth with both hemin and 16-Dehydroprogesterone, as well as the presence of H₂ or pyruvate in the assay. For example, with H₂, activity reached 150 nmol 17-isoprogesterone/mg protein for 16-Dehydroprogesterone reductase and 163 nmol progesterone consumed/mg protein for progesterone reductase. [1] |
| ADME/Pharmacokinetics |
- In cell suspensions of Eubacterium sp. strain 144 under H₂ or pyruvate, 16-Dehydroprogesterone (256 μM) is rapidly reduced to 17-isoprogesterone, which is then further metabolized via reduction of the C4-C5 double bond (ring A) to products that do not absorb UV at 254 nm (presumably 17-isopregnadione and 3-hydroxy derivatives). [1]
- When strain 144 was grown with hemin and 16-Dehydroprogesterone, analysis of culture medium at harvest showed that only 28% (9 nmol/ml) of the initial 16-Dehydroprogesterone (32 μM) remained as 17-isoprogesterone (detectable by UV), indicating extensive metabolism. In contrast, without hemin, 66% (21 nmol/ml) of the steroid was recovered as a mixture of 16α-hydroxyprogesterone, 16-Dehydroprogesterone, and 17-isoprogesterone. [1] - The reduction of 16-Dehydroprogesterone requires exogenous electron donors; H₂ and pyruvate are effective, while glycerol, formate, lactate, and alanine are not. The reaction is dependent on cell protein concentration (linear above 0.5 mg/ml) and time (linear over the first 15 min at 1.3 mg protein/ml). [1] |
| References | |
| Additional Infomation |
16,17-Didehydroprogesterone is a 20-oxosteroid, 3-oxo-Δ(4)steroid, and enone. It is functionally related to progesterone.
- 16-Dehydroprogesterone is an intermediate in the bacterial transformation of 16α-hydroxyprogesterone to 17-isoprogesterone. The reaction is reversible: cell extracts of Eubacterium sp. strain 144 hydrate 16-Dehydroprogesterone back to 16α-hydroxyprogesterone via 16α-dehydroxylase. [1] - In the presence of cysteine (but not Na₂S), 16-Dehydroprogesterone can undergo a non-enzymatic nucleophilic addition to form a water-soluble cysteine-steroid conjugate (tentatively 16S-cysteinylprogesterone). For this reason, Na₂S is used as the reducing agent in the growth medium to prevent this side reaction. [1] - The hemin stimulation of 16-Dehydroprogesterone reductase suggests the involvement of a cytochrome-containing electron transport system that transfers electrons from H₂ or pyruvate to the reductase. This is consistent with observations in related anaerobes such as Eubacterium lentum. [1] |
| Molecular Formula |
C21H28O2
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|---|---|
| Molecular Weight |
312.4458
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| Exact Mass |
312.209
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| CAS # |
1096-38-4
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| PubChem CID |
101964
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| Appearance |
White to off-white solid powder
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| Density |
1.1g/cm3
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| Boiling Point |
461ºC at 760mmHg
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| Melting Point |
185-187 °C(lit.)
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| Flash Point |
171.5ºC
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| Vapour Pressure |
1.11E-08mmHg at 25°C
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| Index of Refraction |
1.557
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| LogP |
4.643
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
23
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| Complexity |
640
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CC(=O)C1=CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CCC4=CC(=O)CC[C@]34C)C
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| InChi Key |
VRRHHTISESGZFN-RKFFNLMFSA-N
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| InChi Code |
InChI=1S/C21H28O2/c1-13(22)17-6-7-18-16-5-4-14-12-15(23)8-10-20(14,2)19(16)9-11-21(17,18)3/h6,12,16,18-19H,4-5,7-11H2,1-3H3/t16-,18-,19-,20-,21+/m0/s1
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| Chemical Name |
(8R,9S,10R,13S,14S)-17-acetyl-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~20 mg/mL (~64.01 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.66 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.66 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: 2 mg/mL (6.40 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2005 mL | 16.0026 mL | 32.0051 mL | |
| 5 mM | 0.6401 mL | 3.2005 mL | 6.4010 mL | |
| 10 mM | 0.3201 mL | 1.6003 mL | 3.2005 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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