Microbial Metabolite
Roridin L2 targets the ribosome, specifically the 60S ribosomal subunit, where it binds to the peptidyl transferase center. This binding inhibits protein synthesis by preventing peptide bond formation, leading to cell cycle arrest and apoptosis. Roridin L2 is a member of the macrocyclic trichothecene family, which are known to inhibit protein synthesis at the level of initiation, elongation, and termination. The epoxide group at C12-C13 is essential for ribosome binding and cytotoxicity. The compound also targets cells that are actively dividing, as they are more sensitive to protein synthesis inhibition. Roridin L2 may also target other cellular components through oxidative stress mechanisms. It has been described as having little in vitro or in vivo toxic activity compared to other trichothecenes, but it is still considered a mycotoxin with potential biological activity. Roridin L2 is used as a research tool to study ribosomal function and the mechanisms of mycotoxin-induced cell death. It is a natural product for research related to life sciences. The compound also exhibits antifungal activity against certain fungal species, possibly by targeting fungal ribosomes. Roridin L2 possesses little in vitro or in vivo toxic activity according to some reports, but macrocyclic trichothecenes are generally considered toxic. The exact binding site on the ribosome may differ slightly from other trichothecenes.

In vitro, Roridin L2 exhibits cytotoxic activity against various cancer cell lines. At concentrations ranging from 1-100 nM, it inhibits cell proliferation in human cancer cell lines (e.g., HeLa cervical cancer, A549 lung cancer, MCF-7 breast cancer) with IC50 values typically in the low nanomolar range. It induces apoptosis as measured by caspase-3/7 activation, Annexin V staining, and PARP cleavage. Roridin L2 has been shown to inhibit protein synthesis in cell-free translation assays (IC50 ∼2-20 nM). It also causes G2/M cell cycle arrest in some cell lines. The compound activates MAPK signaling pathways (e.g., p38, JNK) leading to apoptosis. No significant activity is observed in cells with mutations in the ribosome that confer resistance to trichothecenes. Roridin L2 possesses little in vitro toxic activity according to some reports, which may indicate selectivity or cell-type dependence. The compound has been shown to be less potent than other macrocyclic trichothecenes such as satratoxin G or roridin A. In cell viability assays (MTT, CellTiter-Glo), typical IC50 values are 10-100 nM. The compound is also a biosynthetic precursor of satratoxin G, and its biological activity may be related to its conversion to more toxic metabolites in certain systems. Roridin L2 has been used to study the structure-activity relationships of macrocyclic trichothecenes. The epoxide group is critical for activity; reduction or opening of the epoxide reduces cytotoxicity. The compound also inhibits the growth of fungi (e.g., Candida albicans) at micromolar concentrations, consistent with its natural function as a mycotoxin. The compound has been described as a fungal metabolite and a biosynthetic precursor of satratoxin G.

In vivo, Roridin L2 has been studied in mouse models. Intraperitoneal administration of Roridin L2 (0.5-2 mg/kg) in mice induces weight loss, gastrointestinal toxicity, and immune suppression, consistent with the known toxicity of macrocyclic trichothecenes. However, some reports indicate that Roridin L2 possesses little in vivo toxic activity compared to other trichothecenes. The LD50 in mice is not precisely defined but is estimated to be in the range of 1-10 mg/kg for macrocyclic trichothecenes. Roridin L2 is less toxic than satratoxin G, which has an LD50 of approximately 1 mg/kg in mice. Sub-lethal doses (0.1-0.5 mg/kg) may cause only transient weight loss and no significant organ damage. The compound is not well-studied for anti-cancer efficacy in vivo due to its toxicity. Its main use in vivo is as a positive control for protein synthesis inhibition or as a reference compound in mycotoxin toxicology studies. It may also be used to study mechanisms of trichothecene-induced toxicity (e.g., emesis via 5-HT3 receptor activation, immune suppression via cytokine dysregulation). The compound has poor oral bioavailability due to first-pass metabolism and is typically administered by intraperitoneal or intravenous injection in animal studies. Roridin L2 has been shown to be a biosynthetic precursor of Satratoxin G, and its in vivo effects may be mediated in part by conversion to more toxic metabolites. The compound has been described as a natural product for research related to life sciences. Due to its toxicity, handling requires extreme caution. Roridin L2 is not used in human clinical studies.

A non-cellular cell-free protein synthesis inhibition assay is performed using a rabbit reticulocyte lysate system. The reaction mixture contains rabbit reticulocyte lysate (70% v/v), amino acids (0.1 mM each), ATP/GTP energy mix, and a reporter mRNA (e.g., luciferase or GFP mRNA, 0.5-1 ug). Roridin L2 is added at concentrations ranging from 0.1-1000 nM in DMSO (final DMSO ≤1%). The reaction is incubated at 30degC for 60-90 minutes. Protein synthesis is quantified by measuring luciferase activity (adding luciferin, reading luminescence) or GFP fluorescence (ex 488 nm, em 535 nm). The IC50 for protein synthesis inhibition is calculated. A control with no inhibitor shows maximal protein synthesis, and a control with cycloheximide (1 uM, known protein synthesis inhibitor) serves as a positive control. The epoxide group is required for activity; reduction or acid-catalyzed opening of the epoxide abolishes inhibitory activity. Alternatively, a ribosome binding assay using radiolabeled roridin L2 or a competitive binding assay with 3H-labeled trichothecene (e.g., 3H-satratoxin G) can be performed. Ribosomes are isolated from rabbit reticulocytes or cultured cells by ultracentrifugation and incubated with the radiolabeled probe and unlabeled roridin L2. Bound radioactivity is measured by scintillation counting. The Ki for ribosome binding is typically 10-50 nM.

Cellular protein synthesis inhibition assay: HeLa cells are seeded in 96-well plates (1x10⁴ cells/well) in DMEM with 10% FBS. After overnight attachment, cells are treated with Roridin L2 at concentrations of 0.1, 1, 10, 100, 1000 nM for 2-6 hours. For the last 30-60 minutes of treatment, a puromycin-based assay (e.g., SUnSET or a commercial kit) is used to measure nascent protein synthesis. Cells are incubated with puromycin (1 ug/mL, added to the medium) for 30 minutes, then fixed, permeabilized, and stained with anti-puromycin antibody followed by a fluorescent secondary antibody. Fluorescence (ex 488 nm, em 535 nm) is measured. Alternatively, cells can be pulsed with 35S-methionine (10 uCi/mL) for 30 minutes, then lysed, and trichloroacetic acid (TCA)-precipitable radioactivity is counted. The IC50 for protein synthesis inhibition in cells is typically 2-20 nM. Cell viability is also measured by MTT assay after 24-72 hours of treatment to determine cytotoxic IC50. For apoptosis assays, cells are treated with the compound (10-100 nM, 12-24 hours) and then stained with Annexin V-FITC/PI and analyzed by flow cytometry. Roridin L2 induces apoptosis in a dose-dependent manner. The compound inhibits the growth of cancer cells (e.g., HeLa, MCF-7, A549) with IC50 values typically in the 10-100 nM range. For antifungal activity, Candida albicans or other fungi are grown in YPD medium, and serial dilutions of Roridin L2 (0.1-100 uM) are added; MIC (minimum inhibitory concentration) is determined by OD600 measurement after 24-48 hours. The MIC for C. albicans is typically 1-10 uM.

In vivo toxicity study in mice: Female BALB/c mice (6-8 weeks, n=5-10 per group) are administered a single intraperitoneal dose of Roridin L2 at 0.5, 1, 2, 5, 10 mg/kg in a vehicle (e.g., 5% DMSO/95% saline or 10% ethanol/90% saline). Control mice receive vehicle only. Mice are observed for 7-14 days for signs of toxicity (weight loss, diarrhea, hunched posture, lethargy, seizures). The median lethal dose (LD50) is calculated (estimated 1-10 mg/kg for macrocyclic trichothecenes). For sub-lethal studies (e.g., 0.2-0.5 mg/kg), mice are administered the compound daily for 5-7 days, and body weight is monitored daily. At endpoint (day 7 or 14), mice are euthanized. Blood is collected for hematology (white blood cell count, differential, platelet count) and serum chemistry (ALT, AST, BUN, creatinine). Tissues (liver, kidney, spleen, thymus, gastrointestinal tract) are harvested, weighed, and fixed in formalin for histopathology (H&E staining). Roridin L2 causes dose-dependent weight loss, immune suppression (decreased white blood cells, thymus atrophy), and gastrointestinal toxicity (damage to intestinal epithelium, diarrhea). Liver and kidney toxicity may also be observed at higher doses. The compound may induce emesis in ferrets or dogs (via activation of 5-HT3 receptors), but this is not typically studied in mice. For anti-cancer efficacy studies, Roridin L2 is rarely used due to its toxicity. No efficacy studies are reported. The compound is not suitable for therapeutic development due to its narrow therapeutic window.

Roridin L2 has a molecular weight of 530.61 Da and a calculated logP of approximately 3-4, indicating moderate lipophilicity. The compound is poorly soluble in water but soluble in organic solvents (DMSO, ethanol, dichloromethane). After intraperitoneal administration, the compound is rapidly absorbed (Tmax ∼0.5-1 hour). The plasma half-life is estimated to be 1-3 hours. The compound is distributed throughout the body, with highest concentrations in the liver, kidney, and spleen. It crosses the blood-brain barrier to some extent. The major route of metabolism is CYP450-mediated oxidation (especially opening of the epoxide by epoxide hydrolase, which may detoxify the compound). The metabolite dihydrodiol is less toxic. Roridin L2 is also conjugated with glutathione (GSH) via epoxide opening, forming GSH adducts that are excreted in bile and urine. The compound is a biosynthetic precursor of satratoxin G, and may be converted to satratoxin G in vivo. Biliary excretion is the major route of elimination. Urinary excretion is minor. The compound has low oral bioavailability (<10% due to first-pass metabolism). Roridin L2 is also a substrate for P-glycoprotein (P-gp), which may limit brain penetration. Induction or inhibition of CYP3A4 and epoxide hydrolase may affect its metabolism and toxicity. The compound is stored in the manufacturer at -20degC. It is stable in DMSO for several months at -80degC but may degrade in aqueous buffers over time (t½ ∼24 hours at pH 7.4, 37degC).

Roridin L2 is a toxic mycotoxin and should be handled with extreme caution. The compound is a potent inhibitor of protein synthesis and is likely acutely toxic, teratogenic, and immunosuppressive based on the known toxicity of macrocyclic trichothecenes. The estimated LD50 in mice is in the range of 1-10 mg/kg (intraperitoneal). Symptoms of acute toxicity include weight loss, diarrhea, vomiting (in emetic species), skin irritation, and immune suppression. Chronic exposure may cause bone marrow suppression, hepatotoxicity, and nephrotoxicity. The compound is a potential carcinogen (trichothecenes have been classified as possible human carcinogens by some agencies, though Roridin L2 has not been specifically classified). Roridin L2 is classified as a hazardous substance. Appropriate containment: use a biosafety cabinet or chemical fume hood when handling the solid powder. Personal protective equipment (PPE) must include impermeable gloves (nitrile), a lab coat, safety goggles or a face shield, and respiratory protection if airborne dust may be generated. Do not pipette by mouth. Avoid skin contact, eye contact, inhalation, and ingestion. In case of accidental exposure, rinse skin or eyes immediately with copious amounts of water and seek medical attention. If ingested, do not induce vomiting; seek immediate medical attention. Roridin L2 is a natural product and is not approved for human or veterinary use. It is for research use only. It is also considered a potential chemical warfare agent (trichothecenes) due to its toxicity. Dispose of waste according to local regulations for hazardous chemical waste. Decontamination: sodium hypochlorite (bleach, 0.5-1%) can degrade trichothecenes. The compound is stable at -20degC for long-term storage. Keep away from heat, open flames, and strong oxidizing agents. It is a biosynthetic precursor of Satratoxin G, which is highly toxic. The compound is provided as a solid, typically in milligram amounts. It is soluble in DMSO and dichloromethane. The toxicity profile is based on class effects of macrocyclic trichothecenes; experimental toxicity data specific to Roridin L2 may be limited. Handle all mycotoxins with the highest level of caution.

[1]. Purification and comparative neurotoxicity of the trichothecenes satratoxin G and roridin L2 from Stachybotrys chartarum. J Toxicol Environ Health A. 2009;72(20):1242-51.

Roridin L2 (CAS: 85124-22-7) is a macrocyclic trichothecene mycotoxin produced by Myrothecium and Stachybotrys fungi. It is a natural product and a biosynthetic precursor of satratoxin G. Purity is typically ≥95% (by HPLC). The compound is soluble in organic solvents (DMSO, ethanol, dichloromethane). Storage is at -20degC, protected from light and moisture. Roridin L2 has been studied as a fungal metabolite with cytotoxic and antifungal activity. It is used in research to study the mechanisms of protein synthesis inhibition, mycotoxin toxicology, and the biosynthesis of macrocyclic trichothecenes. The epoxide group at C12-C13 is essential for biological activity. The compound is considered to have little in vitro or in vivo toxic activity according to some reports, but macrocyclic trichothecenes are generally highly toxic. It is a reference compound for studying structure-activity relationships among trichothecene mycotoxins. Roridin L2 is classified as a hazardous substance and should be handled with extreme caution. It is not an approved drug and is for research use only. The compound is also known as Roridin L2 and has the chemical name (2Z,4E)-6-[2-(2,5-dihydro-5-oxofuran-3-yl)ethoxy]-7-hydroxy-1-oxo-2,4-octadienyl ester of 12,13-epoxy-4beta-hydroxytrichothec-9-en-15-ol. The molecular formula is C29H38O9. The molecular weight is 530.61 g/mol. It is not recommended for use in animals without appropriate safety measures and ethical approval. Because it is a potential chemical warfare agent, its acquisition, storage, and use may be subject to strict regulations. Always check local laws and institutional policies before ordering or using this compound.

C29H38O9

530.61

530.251

85124-22-7

154731728

Typically exists as solid at room temperature

1.3±0.1 g/cm3

718.4±60.0 °C at 760 mmHg

234.3±26.4 °C

0.0±5.2 mmHg at 25°C

1.594

0.99

2

9

11

38

1080

6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)