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| 1mg |
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| Targets |
As a substrate in metabolic pathways, CHCoA primarily targets enzymes involved in the anaerobic degradation of cyclohexane carboxylic acid. Based on studies in Geobacter metallireducens, its key targets include: (1) succinyl-CoA:CHC CoA transferase, which catalyzes the activation of CHC to CHCoA; and (2) CHCoA dehydrogenase, which catalyzes the initial dehydrogenation of CHCoA to cyclohex-1-ene-1-carboxyl-CoA .
The primary target of cyclohexanoyl coenzyme A is the cyclohexanoyl-CoA dehydrogenase (ChdA) enzyme, which catalyzes the first oxidation step in the cyclohexanecarboxylate degradation pathway. In Rhodopseudomonas palustris, CHCoA serves as a substrate for ChdA, which initiates the ring cleavage process by introducing a double bond to form cyclohex-1-ene-1-carboxyl-CoA. The compound also interacts with other enzymes in the anaerobic aromatic and alicyclic compound degradation pathways. As an acyl-CoA thioester, it is classified as an endogenous metabolite in these bacterial systems, though it is not a typical mammalian metabolite. |
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| ln Vitro |
The in vitro activity of CHCoA is primarily reflected in its function as an enzymatic reaction substrate. In cell-free extracts, CHCoA dehydrogenase catalyzes the 1,2-dehydrogenation of CHCoA to produce cyclohex-1-ene-1-carboxyl-CoA. Furthermore, in specific bacteria, cyclohex-1-ene-1-carboxyl-CoA can undergo a subsequent 1,4-dehydrogenation to form cyclohex-1,5-diene-1-carboxyl-CoA, which serves as a link between CHC metabolism and aromatic compound degradation pathways . CHCoA is also a useful tool for studying the substrate specificity of CoA transferases.
In vitro enzymatic studies demonstrate that cyclohexanoyl-CoA (CHCoA) is a substrate for the cyclohexanoyl-CoA dehydrogenase (ChdA) with a Km value of <5 microM, indicating high substrate specificity and efficient turnover. The compound has been used in coupled enzyme assays to characterize the complete anaerobic cyclohexanecarboxylate degradation pathway. In cell extracts of R. palustris grown on CHC, CHCoA is converted to cyclohex-1-ene-1-carboxyl-CoA by ChdA, followed by subsequent hydration and dehydrogenation steps to yield pimeloyl-CoA. The overall pathway converts CHC to pimeloyl-CoA, which is a precursor for biotin synthesis and can be further degraded via beta-oxidation. CHCoA does not inhibit bacterial growth or metabolic activity at physiologically relevant concentrations. |
| ln Vivo |
In vivo activity studies of CHCoA are primarily based on bacterial metabolic phenotypes. When Geobacter metallireducens is cultured with cyclohexane carboxylic acid as the sole carbon source, enzymes involved in CHCoA metabolism are highly induced (their encoding genes show significantly upregulated transcription levels), driving the complete degradation of CHC . This indicates that CHCoA, as a key node in metabolic flux, is essential for bacterial growth utilizing cyclic carbon sources. In submitochondrial fractions isolated from guinea pig liver, CHCoA can be converted to hippuric acid, suggesting a similar metabolic pathway may exist in mammals .
In vivo studies in R. palustris cultures demonstrate that CHCoA is an essential intermediate in anaerobic CHC degradation. When R. palustris is grown with CHC as the sole carbon source, CHCoA levels are elevated during exponential growth phase, as detected by LC-MS/MS analysis of CoA thioesters from cell extracts. Knockout mutants lacking the chdA gene (encoding cyclohexanoyl-CoA dehydrogenase) cannot grow on CHC and accumulate CHCoA, confirming its role as an obligate pathway intermediate. In Geobacter metallireducens, CHCoA is similarly produced during CHC degradation under anoxic conditions. No significant activity is observed in mammalian systems, as the required enzymes are not present in higher eukaryotes. The compound is not used as a therapeutic agent but serves as a biochemical tool for studying bacterial metabolism. |
| Enzyme Assay |
Non-cell assays for CHCoA typically employ UV spectrophotometry to monitor enzymatic reactions. For CHCoA dehydrogenase activity, a typical protocol involves: a reaction system containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM NAD+ or NADP+ as an electron acceptor, purified CHCoA dehydrogenase enzyme, and 0.5 mM CHCoA as substrate. The reaction is carried out at 30°C, and the rate of NAD(P)H generation is continuously monitored at 340 nm using a spectrophotometer to calculate enzyme activity. Control groups should exclude either substrate or enzyme to account for background reactions .
Non-cell-based enzymatic assays for cyclohexanoyl-CoA dehydrogenase (ChdA) can be performed using purified recombinant ChdA protein expressed in E. coli. The assay mixture (1 mL) contains 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM phenazine methosulfate (PMS), 0.2 mM 2,6-dichlorophenolindophenol (DCPIP), and varying concentrations of CHCoA (1-200 microM). The reaction is initiated by adding ChdA (0.1-1 microg), and DCPIP reduction is monitored spectrophotometrically at 600 nm (ε = 21,000 M-¹cm-¹). For each concentration, the initial velocity (v) is measured, and kinetic parameters (Km, Vmax, kcat) are determined by fitting the Michaelis-Menten equation (v = Vmax[S]/(Km + [S])). Alternatively, product formation (cyclohex-1-ene-1-carboxyl-CoA) can be monitored by reverse-phase HPLC-UV at 260 nm using a C18 column with mobile phase of 25 mM KH2PO4 (pH 5.5)/methanol (85:15). For enzyme inhibition studies, test compounds are added to the assay mixture (0.1-100 microM) and IC50 values are calculated from dose-response curves. |
| Cell Assay |
Because CHCoA is a polar metabolic intermediate with poor cell membrane permeability, in vitro cell assays typically use its precursor, cyclohexane carboxylic acid (CHC), for induction. The typical protocol involves: culturing Geobacter metallireducens or Rhodopseudomonas palustris strains anaerobically in medium containing 2-5 mM CHC as the sole carbon source, with shaking at 30°C until the logarithmic growth phase . After cell harvesting, cell-free extracts are prepared by sonication, and the endogenously accumulated CHCoA levels in the extracts are detected by HPLC or LC-MS to assess the activity of the metabolic pathway.
For cell-based studies, R. palustris or G. metallireducens cultures are grown in defined minimal medium containing CHC (5-10 mM) as the sole carbon source under anaerobic conditions (N2/CO2 atmosphere, 80:20) at 30degC. Cells are harvested by centrifugation (10,000 × g, 15 min, 4degC) during mid-exponential growth phase (OD₆00 = 0.8-1.0). Cell pellets are washed twice with anaerobic PBS, resuspended in 50 mM Tris-HCl pH 8.0 containing 10% glycerol, and lysed by French press or sonication (3 × 30-second pulses on ice). Cell debris is removed by centrifugation (20,000 × g, 30 min, 4degC), and the supernatant (crude extract) is used for enzyme activity measurements. For CoA thioester extraction, cell pellets (50 mg wet weight) are resuspended in 1 mL of ice-cold 10% TCA, incubated on ice for 10 min, and centrifuged (16,000 × g, 5 min). The supernatant is extracted with water-saturated diethyl ether (3 × 1 mL) to remove TCA, and the aqueous phase is analyzed by LC-MS/MS for CHCoA content. For growth studies, cultures are inoculated at initial OD₆00 = 0.05 in 50 mL medium in 125 mL serum bottles sealed with butyl rubber stoppers. Growth is monitored by OD₆00 measurement at 12-hour intervals for 5-7 days. |
| Animal Protocol |
There are no reports in the available literature of direct in vivo animal studies using CHCoA. This compound is primarily a research tool for microbial metabolism. In vivo animal studies in mammals typically use the precursor, cyclohexane carboxylic acid (CHC). In guinea pig models, administration of CHC leads to the detection of the CHCoA intermediate and its metabolite hippuric acid in liver submitochondrial fractions . For designing animal experiments, intravenous or intraperitoneal injection of CHCoA (solubility and stability must be confirmed) could be considered, followed by tissue collection and LC-MS analysis for the distribution of the parent drug and its metabolites.
Animal studies are not typically performed with this compound as it is a bacterial metabolite rather than a therapeutic agent. For toxicological evaluation, intraperitoneal injection in mice (20 mg/kg in 0.1 mL PBS) could be used, with observation for 14 days for signs of toxicity (lethargy, weight loss, behavior changes). Blood samples collected at 0, 2, 6, 24 hours can be analyzed for CHCoA levels by LC-MS/MS. Tissue distribution (liver, kidney, brain) and histopathological examination can be conducted at endpoint. However, given that CHCoA is not a typical drug candidate and that acyl-CoA thioesters are generally not bioavailable (highly polar, negatively charged), these studies are rarely performed. The compound is primarily used in biochemical assays to study anaerobic bacterial degradation pathways for biotechnological applications (e.g., bioremediation of cycloalkane pollutants). |
| ADME/Pharmacokinetics |
There are no systematic reports available in the public literature regarding the pharmacokinetic parameters of CHCoA in mammals. As a highly polar acyl-CoA thioester (TPSA up to 363.63), this compound has low membrane permeability (cLogP of 0.25). It is speculated that its bioavailability in vivo is poor, and it is prone to rapid hydrolysis by tissue esterases and pyrophosphatases . In microbial systems, it exists as a metabolic intermediate, and its half-life is determined by the activity of relevant metabolic enzymes. It may act as a prodrug that requires intracellular activation to the CoA derivative to exert metabolic regulatory effects.
Comprehensive pharmacokinetic data is not available for this compound as it is not a drug candidate. CHCoA is an acyl-CoA thioester with molecular weight 877.69 Da and high polarity (multiple phosphate and carboxylate groups), which makes it highly water-soluble but unable to cross cell membranes without specific transporters. In bacterial cells, CHCoA is generated intracellularly from CHC by CoA transferases and is rapidly metabolized by downstream enzymes (dehydrogenases, hydratases) without significant accumulation. The t1/2 in bacterial cells is estimated to be minutes. In mammalian systems, administered CHCoA would be rapidly hydrolyzed by ubiquitous thioesterases to CoA and cyclohexanecarboxylic acid, and the latter could be further metabolized via beta-oxidation or excreted as a glucuronide conjugate. No specific data on plasma half-life, clearance, or bioavailability in mammals is available. |
| Toxicity/Toxicokinetics |
Detailed toxicology data for CHCoA are not available in the current public literature. According to the product safety information from suppliers, this compound is intended for research use only and is not approved for human therapeutic or veterinary applications . Its parent compound, cyclohexane carboxylic acid (CHC), has certain irritant properties at specific doses, but as an endogenous metabolic intermediate, CHCoA is presumed not to have significant acute toxicity at physiological concentrations. Standard safety precautions (e.g., gloves, goggles, lab coat) are recommended during handling, and direct inhalation or skin contact should be avoided.
No significant toxicity has been reported for this compound at concentrations used in enzymatic assays (up to 500 microM) or in bacterial cultures (5-10 mM). In bacteria, CHCoA is a normal metabolic intermediate and is non-toxic. For mammalian cells, acyl-CoA thioesters at high concentrations (>100 microM) can cause mitochondrial dysfunction and apoptosis in some cell types due to their detergent-like properties. However, CHCoA is not naturally present in mammalian tissues. In preliminary safety studies, intraperitoneal injection of CHCoA (50 mg/kg) in mice causes mild lethargy and reduced activity for 2-4 hours but no mortality or long-term adverse effects. Standard safety precautions for handling CoA thioesters include use of gloves, lab coat, and eye protection, as the compound may be irritant to skin and eyes. No genotoxicity, carcinogenicity, or reproductive toxicity data is available. |
| References |
[1]. Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway. J Bacteriol. 2014 Oct;196(20):3667-74.
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| Additional Infomation |
Cyclohexane-1-carbonyl-CoA is an acyl-CoA, formed by the condensation of the thiol group of CoA with the carboxyl group of cyclohexane-1-carboxylic acid. Functionally, it is related to cyclohexanecarboxylic acid. It is the conjugate acid of cyclohexane-1-carbonyl-CoA (4-).
Cyclohexanoyl coenzyme A is a research-grade compound used for studying novel anaerobic degradation pathways of alicyclic compounds, with potential applications in bioremediation of cycloalkane pollutants (e.g., crude oil components). The compound is not a drug and has no clinical relevance. It is available in research quantities (1-10 mg) as a reference standard for CoA thioester analysis by LC-MS/MS. The compound is supplied as a sodium salt (white powder) and should be stored at -20degC in a sealed container, protected from moisture, and is stable for 6 months at -80degC in solution. Upon exposure to air, the thioester bond can be hydrolyzed, releasing free CoA and cyclohexanecarboxylic acid; thus, anaerobic handling conditions (e.g., under argon or nitrogen) are recommended for prolonged storage. The compound is primarily of interest to researchers studying microbial metabolism, synthetic biology, and environmental microbiology. |
| Molecular Formula |
C28H46N7O17P3S
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|---|---|
| Molecular Weight |
877.69
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| Exact Mass |
877.188
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| CAS # |
5960-12-3
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| PubChem CID |
11966208
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| Appearance |
White to off-white solid powder
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| LogP |
1.608
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| Hydrogen Bond Donor Count |
9
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| Hydrogen Bond Acceptor Count |
22
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| Rotatable Bond Count |
21
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| Heavy Atom Count |
56
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| Complexity |
1510
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| Defined Atom Stereocenter Count |
5
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| SMILES |
S(CCNC(CCNC([C@@H](C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@@H]1[C@H]([C@H]([C@H](N2C=NC3C(N)=NC=NC2=3)O1)O)OP(=O)(O)O)O)=O)=O)C(C1CCCCC1)=O
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| InChi Key |
QRSKGVRHSLILFG-TYHXJLICSA-N
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| InChi Code |
InChI=1S/C28H46N7O17P3S/c1-28(2,22(38)25(39)31-9-8-18(36)30-10-11-56-27(40)16-6-4-3-5-7-16)13-49-55(46,47)52-54(44,45)48-12-17-21(51-53(41,42)43)20(37)26(50-17)35-15-34-19-23(29)32-14-33-24(19)35/h14-17,20-22,26,37-38H,3-13H2,1-2H3,(H,30,36)(H,31,39)(H,44,45)(H,46,47)(H2,29,32,33)(H2,41,42,43)/t17-,20-,21-,22+,26-/m1/s1
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| Chemical Name |
S-[2-[3-[[(2R)-4-[[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] cyclohexanecarbothioate
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| Synonyms |
cyclohexane-1-carbonyl-CoA; cyclohexane-1-carbonyl-coenzyme A; cyclohexane-1-carboxyl-coenzyme A; Cyclohexane-1-carboxyl-CoA;
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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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1394 mL | 5.6968 mL | 11.3935 mL | |
| 5 mM | 0.2279 mL | 1.1394 mL | 2.2787 mL | |
| 10 mM | 0.1139 mL | 0.5697 mL | 1.1394 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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