Synthesis of derivatives of ferricrocin, a strong iron-binding peptide, useful for preparation of an ELISA assay.

摘要

Iron is an essential micronutrient required by living organisms. Many microbes secrete low molecular weight compounds called siderophores to obtain iron from the environment. The E-strain mycorrhizal fungi, Wilcoxina spp., produce the siderophore, ferricrocin (FCR) under iron-limiting conditions as part of a high-affinity iron uptake system. The objectives of this dissertation were to synthesize derivatives of FCR that could be used to prepare a protein-FCR conjugate suitable for development of an immunoassay for FCR. FCR-hemisuccinate, FCR-hemiadipate, FCR-hemisuberate, FCR-adipyl-FCR, and FCR-hemiterephthalate were synthesized using Steglich esterification, and their structures were confirmed by LC-MS and/or NMR spectroscopy. The yields for the first three compounds were greater than 50%. FCR-hemisuccinate was activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and reacted with the proteins KLH and BSA. This protein conjugate was injected into rabbits for antibody production. The results from immunoblotting, titer determination, and competitive enzyme-linked immunosorbent assays (ELISA) indicated that the antibodies were specific to the N-acylurea derivative of EDC and had little or no specificity to free FCR. The low specificity of the antibodies to FCR was attributed to the labile character of the ester linkage of FCR-hemisuccinate. LC-MS and GPC studies showed that the ineffectiveness of the FCR-hemisuccinate activation by EDC and of the subsequent amidation led to a low yield for BSA-succinyl-FCR and the formation of the N-acylurea derivative of the EDC-protein conjugates. This dissertation demonstrated the broad application of Steglich esterification for derivatization of carboxyl-containing compounds. It also illustrated several problems when using carbodiimide activation to prepare protein-hapten conjugates and their corresponding antibodies. Different carbodiimides should be used to prepare the protein-hapten conjugate used to make the antibodies needed for an immunoassay to exclude antibody recognition of the N-acylurea derivative of a carbodiimide.