Ethyl 4-Bromobutyrate: Synthesis, Applications, and Role in Zilpaterol ELISA Development

Ethyl 4-Bromobutyrate is a colorless to light yellow clear liquid with a molecular formula of C6H11BrO2. It is widely used as an intermediate for pesticides, medicines and other pharmaceuticals.

Synthesis of ethyl 4-bromobutyrate from γ-butyrolactone

The invention aims to solve the technical problems existing in the prior art, and provides a one-step preparation method of ethyl 4-bromobutyrate. In order to achieve the above object, the present invention adopts the following technical scheme: a one-step preparation method of ethyl 4-bromobutyrate, comprising the steps: Step 1, under the condition of water bath and uniform stirring, add γ-butyrolactone to the container, and control the temperature of the water bath to be 10-30°C; Step 2, slowly feed dry hydrogen bromide gas; Step 3, after the hydrogen bromide gas is passed in, the temperature of the control reaction is 0-50 ° C, and the stirring reaction is continued for 1-3 hours; Step 4, adding absolute ethanol, controlling the constant temperature of the water bath to be 10-90 °C, and continuing to stir for 2-6 hours, the reaction is completed; Step 5: After the reaction is completed, add an appropriate amount of deionized water for washing, stir well, stand for stratification, remove the lower organic phase, add saturated NaHCO3 solution, adjust the pH to 7.0, stand for stratification, remove the lower organic phase, pass through Wash with deionized water, stir well, stand for stratification, and separate the organic layer; Step 6, adding a desiccant and drying to obtain ethyl 4-bromobutyrate.[1]

Principle and beneficial effects of the present invention: (1) synthesizing ethyl 4-bromobutyrate by one-step method using γ-butyrolactone as raw material, the amount of hydrogen bromide gas introduced into γ-butyrolactone first is 1.2 times (molar ratio), the best production effect can be obtained when the amount of ethanol added subsequently is 1.06 times (molar ratio). The invention has the advantages of no separation of synthetic intermediates, simple process and easy operation, shortened reaction time, no by-products detected by gas chromatography, high utilization rate of raw materials, product purity as high as 98% or more, yield as high as 93% or more, and no by-products. pollution, meet the requirements of green chemistry, and can meet the needs of high-purity industrial scale production.

Compared with the existing production technology, the present invention adopts γ-butyrolactone as raw material to prepare ethyl 4-bromobutyrate by one-step method, firstly feeding an appropriate amount of dry hydrogen bromide gas and then adding an appropriate amount of anhydrous ethanol in one step. The method obtains ethyl 4-bromobutyrate, which effectively solves the problems of excessive hydrogen bromide gas required in the existing production process, producing a large amount of waste water, waste gas and waste liquid, low yield and low purity. The synthetic process route is simple, the yield is as high as over 93%, and the product purity is over 98%. In addition, the method of the invention has low production cost, simple equipment requirements, green and pollution-free, and is suitable for industrialized large-scale production.

Synthesis from 4-bromobutanoic acid

A solution of 4-bromobutanoic acid (25.0 g, 150 mmol), DMF (5 drops) and oxalyl chloride (17.0 mL, 195.0 mmol) in dichloromethane (250 mL) was stirred at room temperature for 5 hr. The reaction mixture was concentrated under reduced pressure, dichloromethane (200 mL) and ethanol (9.0 mL, 170 mmol) were added to the residue, and the mixture was stirred at room temperature for 15 hr. The reaction mixture was washed with saturated aqueous sodium hydrogencarbonate and saturated brine, and the organic layer was dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (0 - 5% ethyl acetate/hexane) to give ethyl 4-bromobutyrate as a colorless oil (24.5 g, 84%).[2]

A suspension of 3-hydroxybenzonitrile (5.00 g, 42.0 mmol) and 60% sodium hydride (oil dispersion, 2.01 g, 50.3 mmol) in DMF (200 mL) was stirred at room temperature for 30 min, ethyl 4-bromobutanoate (9.82 g, 50.3 mmol) was added thereto, and the mixture was stirred at room temperature for 15 hr. The mixture was allowed to cool to room temperature, and concentrated under reduced pressure. The residue was extracted with ethyl acetate and saturated aqueous ammonium chloride solution. The organic layer was washed with saturated aqueous ammonium chloride solution and saturated brine, dried over sodium sulfate, and concentrated. The obtained residue was purified by silica gel column chromatography (0 - 10% ethyl acetate/hexane) to give ethyl 4-bromobutyrate as a colorless oil (10.2 g, 100%).1 H-NMR (300MHz, CDCl3 ) δ: 1.27 (3H, t, J = 7.2 Hz), 2.13 (2H, tt, J = 6.7, 6.7 Hz), 2.52 (2H, t, J = 7.2Hz), 4.03 (2H, t, J = 6.1 Hz), 4.16 (2H, q, J = 7.2 Hz), 7.08-7.16 (2H, m), 7.21-7.26 (1H, m), 7.32-7.40 (1H, m).

Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol

Zilpaterol is an β-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growth promoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the development of an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in 0.1 M potassium hydroxide. Briefly, 100 mg of zilpaterol HCl was added to 10 mL of 1 N NaOH (pH >10) and the zilpaterol free base was extracted with ethyl acetate (5 × 5 mL). The ethyl acetate layer was dried with anhydrous sodium sulfate and the solvent evaporated with a rotary evaporator. To a 50-mL three-necked, round-bottom flask equipped with a mechanical stirrer, a condenser, and an addition funnel was added 32 mg (0.12 mmol) of zilpaterol freebase (oil) dissolved in 10 mL of acetone. Ethyl 4-bromobutyrate (17.5 μL, 0.12 mmol) and 66 mg (0.48 mmol) of potassium carbonate were added and the reaction was refluxed with stirring overnight.[3]

When developing an immunoassay the hapten must be carefully designed to achieve appropriate specificity. We elected to couple zilpaterol with ethyl 4-bromobutyrate under conditions which ultimately allow the free carboxyl group of the butyrate linker to be activated and coupled to the amino groups of the target protein. Zilpaterol contains three nucleophilic groups (a secondary alcohol, a secondary amine, and the benzimidazole amide) that are potentially capable of displacing the bromine on ethyl 4-bromobutyrate. The secondary alcohol group of zilpaterol would be expected to be considerably less reactive than the aliphatic or benzimidazole nitrogens. The secondary amine would have considerable steric hindrance due to the freely rotating isopropyl group, so the most likely reaction site for zilpaterol and ethyl 4-bromobutyrate would be the secondary amide on the benzimidazole portion of zilpaterol. From a design viewpoint, either the secondary amine or the amide would likely produce a suitable hapten. We believed that in the zilpaterol-conjugated protein, the three-carbon distance between the carboxyl group conjugated to the protein and the zilpaterol molecule would be adequate for efficient coupling and the appropriate exposure of the zilpaterol moiety to promote antibody specificity. The synthesis of the hapten, zilpaterol-butyric acid proved to be routine.

We attempted to identify the actual site of 4-bromobutyrate conjugation to zilpaterol via the use of mass spectrometry. The proposed fragmentation of zilpaterol-butyric acid is shown. In conclusion, using a hapten generated by reacting zilpaterol with ethyl 4-bromobutyrate followed by alkaline hydrolysis an antigen was generated that produced a very specific antibody to zilpaterol. This antibody was used to develop an ELISA that was capable of determining zilpaterol with an IC50 of 3.94 ± 0.48 ng/mL, sufficient sensitivity for residue analysis. The assay was tolerant to low concentrations of organic solvents and demonstrated minimal matrix effects when used to analyze bovine and porcine urine providing the samples were diluted with 1% BSA/PBST.

References

[1]MYJ CHEMICAL - CN114736119, 2022, A

[2]TAKEDA PHARMACEUTICAL - EP1953148, 2008, A1

[3]Shelver, Weilin L, and David J Smith. “Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol.” Journal of agricultural and food chemistry vol. 52,8 (2004): 2159-66.

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