SARS-CoV-2[1]
β-Propiolactone is a direct-acting alkylating agent. Its primary mechanism of action is irreversible alkylation via its highly reactive four-membered lactone ring, which opens to alkylate nucleophilic groups such as carboxyl groups in proteins and hydroxyl groups, but most significantly, the nitrogen atoms in the purine and pyrimidine bases of nucleic acids. This reaction forms stable carboxyethyl adducts, mainly at the N7 position of guanine and the N1 position of adenine, leading to the destruction of genetic material and pathogen inactivation.

For the purification of vaccines, propiolactone (β-propiolactone) can be employed. Following the collection of cells using low-speed centrifugation, Propiolactone (1:1000 v:v) was used to chemically inactivate SARS-CoV. SARS-CoV was incubated with propiolactone for a whole day at 4°C. To hydrolyze any remaining propiolactone, a second incubation is carried out at room temperature. and the vaccine's concentration. after BPL deactivation. To precipitate the inactivated virus, a combination of polyethylene glycol and sodium chloride (PEG-NaCl) was applied. following vaccination and concentration. A combination of polyethylene glycol and sodium chloride (PEG-NaCl) is used to inactivate the virus and precipitate it once propiolactone has been rendered inactive. As a final bacteriostatic agent, 1:10000 v:v propiolactone was applied. In Vero cells, the propiolactone-inactivated virus becomes less contagious [1].

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In vitro, propiolactone is employed as a chemical inactivator for viruses. For SARS-CoV, propiolactone was co-incubated at a ratio of 1:1000 (v:v) for a full day at 4°C to chemically inactivate the virus, followed by a second incubation at room temperature to hydrolyze any remaining propiolactone. In Vero cells, the propiolactone-inactivated virus becomes less contagious. It is also used as a bacteriostatic agent to prepare BPL-inactivated influenza virus vaccine (Flu-BPL).

The influenza A virus was inactivated with propiolactone (β-propiolactone) and injected intramuscularly into mice at a dose of approximately 25 mg total protein. Young BALB/c mice treated with propiolactone inactivation are not fatally affected by SARS. Even while the virus continued to grow in the mice's respiratory system, by day five it had disappeared. After the mice were infected, 1.5 μg of total hemagglutinin protein were produced as a result of propiolactone treatment, which was negative [1].

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In vivo, influenza A virus inactivated with propiolactone was injected intramuscularly into young BALB/c mice at a dose of approximately 25 mg total protein. Mice treated with propiolactone inactivation were not fatally affected by SARS. Although the virus continued to grow in the respiratory system, it had disappeared by day five. Propiolactone treatment resulted in the production of 1.5 μg of total hemagglutinin protein following infection.

For non-cellular in vitro enzyme/receptor binding assays, β-propiolactone is typically tested as an alkylating agent in nucleic acid interaction studies. The compound is incubated with purified DNA or RNA under controlled conditions (e.g., 4°C to 37°C) for specified durations. Following incubation, the formation of carboxyethyl adducts is analyzed using techniques such as mass spectrometry or HPLC to quantify alkylation at specific nucleobase positions, particularly guanine N7 and adenine N1.

For in vitro cellular assays, viruses such as SARS-CoV are propagated in Vero cell cultures. Following cell collection via low-speed centrifugation, propiolactone (1:1000 v:v) is used to chemically inactivate the virus. After inactivation, the virus is precipitated using a combination of polyethylene glycol and sodium chloride (PEG-NaCl). The inactivation efficacy is then assessed by evaluating the loss of viral infectivity in fresh Vero cell cultures.

For in vivo animal studies, the influenza A virus is inactivated with propiolactone and administered intramuscularly to young BALB/c mice at a dose of approximately 25 mg total protein. The protective efficacy is evaluated by subsequent viral challenge, monitoring survival rates, viral titers in the respiratory system, and immune responses such as hemagglutinin protein production.

Absorption, Distribution and Excretion
The LD50 for guinea pig dermal administration is less than 5 mL/kg, indicating good absorption. β-Propiolactone binds to DNA, RNA, and proteins in mouse skin in vivo. Tumor initiation activity is proportional to the degree of DNA binding but independent of RNA or protein binding. The main RNA and DNA binding product is 7-(2-carboxyethyl)guanine. S-2-carboxyethylcysteine…is present in the acid hydrolysis products of proteins… Metabolisms/Metabolites Propiolactone is completely hydrolyzed in aqueous solution after 3 hours; hydrolysis may be faster in the presence of cell debris and cell culture medium. In water, the lactone ring opens at the alkyl and acyl bonds. The degradation products of propiolactone are non-toxic. β-Propiolactone can react with chloride ions to form 3-chloropropionic acid, especially in plasma.
β-hydroxypropionic acid, the hydrolysis product of β-propiolactone, did not induce local sarcomas in subcutaneous injection in rats, nor did it induce skin tumors when applied to the skin of mice.
Biological half-life

The half-life of propiolactone in water is 225 minutes.
No relevant reports found; [TDR, page 1048]

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β-Propiolactone is rapidly hydrolyzed in aqueous environments to beta-hydroxypropionic acid, which is non-toxic. Its pharmacokinetic profile is characterized by rapid degradation and short half-life in biological systems, which makes it suitable for virus inactivation applications where residual activity must be eliminated. The compound is typically inactivated by hydrolysis after use to prevent prolonged exposure.

Protein Binding
Propiolactone has a strong protein binding capacity, almost twice that of DNA and RNA. Toxicity Data LC50 (rat) = 25 ppm/6h Interactions Intravenous injection of β-propiolactone alone causes liver necrosis and renal tubular damage, but its toxicity is significantly reduced if it is reacted with proteins before injection. This study investigated the ability of UV-A light (320-400 nm) to induce cell transformation in vitro and to alter chemically induced cell transformation in BALB/c 3T3 cell cultures. UV-A alone induced cell transformation when irradiated with a series of non-toxic doses, and the transformation rate was linearly related to the number of UV-A irradiations. To investigate potential interactions between UV-A and environmental chemical carcinogens, we used a standard initiation/promotion protocol to examine the effect of UV-A irradiation on cell transformation exposed to the direct carcinogen β-propiolactone (an alkylating agent). Cells were treated with a single dose of 2.5 μg/ml β-propiolactone for 24 hours, followed by exposure to 3.0 kJ/m² UV-A light. UV-A irradiation was repeated weekly for 5 weeks. Cells were then fixed, stained, and type III transformation foci were counted in the culture dishes. Weekly UV-A irradiation alone for 5 weeks induced approximately 3 transformation foci per culture dish. β-propiolactone treatment alone induced approximately 1 foci per culture dish (background: 0.17 foci per culture dish). The combined use of both treatments significantly increased the yield of transformation foci per culture dish, and the enhancing effect of UV-A irradiation increased with the number of irradiations (approximately 10 foci per culture dish after a single β-propiolactone irradiation and after 5 UV-A irradiations). These results suggest a synergistic effect between β-propiolactone and subsequent UV-A irradiation in in vitro tumor transformation induction. Research is currently underway to identify compounds that can trap direct-acting carcinogens within the gastrointestinal lumen, thereby preventing these carcinogens from attacking host tissues. Sodium 4-mercaptobenzenesulfonate, a strong nucleophile, has been shown to react rapidly in vitro with the direct-acting carcinogen β-propiolactone. Further studies have demonstrated that sodium 4-mercaptobenzenesulfonate can inhibit mutations in Salmonella Typhimurium TA-100 strain after exposure to β-propiolactone and another direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine. A series of experiments were subsequently conducted to determine whether sodium 4-mercaptobenzenesulfonate could inhibit β-propiolactone-induced carcinogenesis in vivo. In the first experiment, researchers administered sodium 4-mercaptobenzenesulfonate by gavage to female A/J mice 5 minutes before oral administration of β-propiolactone. The results showed that under these conditions, the carcinogenic effects of the forestomach were inhibited. In the second experiment, researchers administered sodium 4-mercaptobenzenesulfonate rectally by gavage 5 minutes before administering β-propiolactone to the rectum of mice. Rectal administration of β-propiolactone induced colorectal adenomatous polyps. Pre-administration of sodium 4-mercaptobenzenesulfonate inhibited the development of these tumors. These results indicate that sodium 4-mercaptobenzenesulfonate has the ability to capture direct carcinogens and inhibit β-propiolactone-induced tumorigenesis. A two-step carcinogenesis protocol was used. SENCAR mice were first induced to develop cancer with 25 μg of 7,12-dimethylbenzo[a]anthracene, and then twice weekly were given either (a) 0.5 mg β-propiolactone or (b) 1 μg fluocinolone acetonide, followed by 0.5 mg β-propiolactone acetonide 30 minutes later. The tumor incidence rate in the group treated with fluocinolone acetonide before β-propiolactone administration was significantly higher than that in the group treated with β-propiolactone alone (p<0.0005). Under these experimental conditions, β-propiolactone alone showed neither pro-cancer activity nor complete carcinogenic activity. These results were unexpected, but the reasons are under investigation. Research has been initiated to identify compounds that can retain directly acting carcinogens in the stomach. Sodium thiosulfate, a strong nucleophile, has been shown in preliminary experiments to inhibit mutations in Salmonella Typhimurium TA100 strain after exposure to the directly acting carcinogens β-propiolactone and styrene oxide. In vitro experiments showed that sodium thiosulfate retains its nucleophilicity within an acidic pH range. Its reaction rate with β-propiolactone at pH 2 is as fast as at pH 7.4. Therefore, sodium thiosulfate possesses the necessary properties to inhibit the carcinogenic effects of electrophilic agents in the stomach. In experiments, sodium thiosulfate was administered by gavage to female A/J mice 5 minutes before oral administration of β-propiolactone. Under these conditions, forestomach tumor formation was inhibited. The data suggest that using nucleophiles to counteract directly acting carcinogens is a potential chemoprevention strategy.

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Non-human toxicity values
Rat inhalation LC50: 250 ppm/30 min
Rat inhalation LC50: 25 ppm/6 h
Intravenous LD50 in young rats: 225 ± 55 mg/kg, scoring after 24 hours
Dermal LD50 in guinea pigs: < 5 ml/kg

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β-Propiolactone is a recognized carcinogen and must be handled with extreme care. It is toxic by inhalation, in contact with skin, and if swallowed. It causes severe skin burns and eye damage. Appropriate personal protective equipment (PPE) and engineering controls are mandatory. A crucial safety step in any protocol is the hydrolysis of residual BPL to non-toxic beta-hydroxy-propionic acid after the inactivation process is complete.

[1]. Immunogenicity and protective efficacy in mice and hamsters of a β-propiolactone inactivated whole virus SARS-CoV vaccine. Viral Immunol. 2010 Oct;23(5):509-19.

[2]. Anti-SARS-CoV-2 IgG antibody response among Indian COVID-19 patients using β-propiolactone-inactivated, whole virus-based indirect ELISA. J Virol Methods. 2021 Jan;287:113996.

Drug Warnings
Since skin cancer is considered the primary concern regarding BPL toxicity, contact with liquid BPL should be avoided. Pharmacodynamics Under optimal conditions, propiolactone is approximately 25 times more active as a gas-phase disinfectant than formaldehyde, approximately 4000 times more active than ethylene oxide, and approximately 50,000 times more active than methyl bromide. It has been shown to be mutagenic, exerting its effects by inducing cell transformation, chromosomal aberrations, and chromatin exchange. Propiolactone has been shown to be mutagenic in both somatic and germ cells.

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Propiolactone is used as a viral chemical inactivator that causes the infectious inactivation of viruses while maintaining immunogenicity, as it directly acts on viral nucleic acids without destroying capsid proteins. It has been used in the preparation of inactivated vaccines including those for Influenza, SARS-CoV, and SARS-CoV-2. As a sporicidal agent, it is also employed for sterilization of sensitive biological materials such as tissue grafts, blood plasma, and surgical instruments where traditional heat methods are not feasible.

C3H4O2

72.06

72.021

57-57-8

25037-58-5

2365

Colorless to light yellow liquid

1.2±0.1 g/cm3

162.0±0.0 °C at 760 mmHg

-27 °F (EPA, 1998) ; -33.4ºC ; -33.4 °C ; -33.4 °C ; -28 °F ; -33.4 °C ; -28 °F

35.0±16.1 °C

2.2±0.3 mmHg at 25°C

1.445

-1.33

0

2

0

5

57.9

0

O1C(C([H])([H])C1([H])[H])=O

VEZXCJBBBCKRPI-UHFFFAOYSA-N

InChI=1S/C3H4O2/c4-3-1-2-5-3/h1-2H2

oxetan-2-one

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.)