Carboxyl Derivatives of Chloramphenicol with Stable Linkers in the Side Chain and Their Immunoreactive Conjugates with Horseradish Peroxidase

Objective: In order to ensure the biosafety of food products, they are screened for the presence of the antibiotic chloramphenicol, which is prohibited for veterinary use. For rapid testing, various types of immunoassay are often used, including enzyme-linked immunosorbent assay (ELISA). Chloramphenicol hemisuccinate is an ester of succinic acid. It is often used to obtain the enzyme-labeled conjugate required in ELISA. Long-term storage of this compound in solution leads to hydrolysis of the ester bond, so chloramphenicol hemisuccinate conjugates are fairly labile compounds. The aim of the work is to obtain new derivatives of chloramphenicol at the C3 OH group, the conjugates of which with the enzyme would have greater stability over a long period of storage in the form of a dilute solution. Methods: The new ester and ether at the C3 OH group of chloramphenicol, as well as their conjugates with horseradish peroxidase, were synthesized. The compounds were studied in direct competitive ELISA. Storage at high temperatures or in the Tris-HCl buffer pH 8.7 was used to determine the stability of the conjugates. Results and Discussion: The first derivative of chloramphenicol was obtained by acylation of the C3 OH group with trans-methylcyclohexanedicarboxylic acid anhydride. The product of the reaction is an ester, the hydrolysis of which can be sterically hindered by the cyclohexane ring. Chloramphenicol ether was obtained by adding 4-bromomethylphenylpropionic acid tert-butyl ester to the C3 OH group of the antibiotic. The design of the alkylating reagent allowed carrying out the reaction with chloramphenicol under mild conditions, deprotecting the introduced carboxyl group without the risk of reactions occurring on other functional groups of the antibiotic, and ensuring the increased distance between the chloramphenicol benzene ring and the polypeptide chain of the protein. Enzyme conjugates were obtained using the new derivatives of chloramphenicol and used as labels in ELISA. Storage experiments revealed significant differences in the stability of the conjugates and showed that the most stable was the one in which the antibiotic was tied to the linker by the ether bond. We found that the use of this conjugate in ELISA allowed the determination of chloramphenicol in milk with a limit of quantification of 0.04 μg/kg, the recovery of chloramphenicol additive was 105%. Conclusions: A chloramphenicol conjugate in which the antibiotic is bound to a linker by an ether bond exhibits high stability when stored in dilute solution. Testing of this new conjugate in the ELISA yielded results showing the achievement of the practically important goal of the study