Production and Purification of N‐Hydroxy Succinyl Cadaverine (HSC) By Synthetic and Biosynthetic Methods

Abstract

Optimum conditions for the production and purification of N‐Hydroxy Succinyl Cadaverine, HSC, were investigated. HSC is not commercially available and is essential for research with an enzyme known as DesD. This enzyme will allow HSC to be trimerized and macrocyclized into a siderophore. The siderophore is capable of absorbing iron in the bloodstream that antibiotic‐resistant bacteria use as a life‐sustaining nutrient. Small‐scale organic synthesis was involved in the process of producing the precursor to HSC, known as SC. SC, succinyl cadaverine, is non‐hydroxylated HSC. A deprotection of Boc‐protected succinyl cadaverine was performed. Regular phase thin‐layer chromatography (TLC) was used to check reaction progress while reverse phase TLC was used to determine solvent conditions for purification of SC. Previous attempts to create SC yielded orange‐red puddles of oil. Reaction conditions were changed to theoretically yield white crystals, which indicates purity. After decreasing the equivalencies of TFA, changing the solvent of the reaction, and increasing the run time, white crystals were achieved. 21.5 milligrams of SC were produced in total. The appearance of the crystals increased confidence that the product was fairly pure. Regardless, purification was necessary. The solvent condition for purification of SC was determined to be 100% dichloromethane. This solvent condition yielded a Rf of ~0.5 on a reverse‐phase TLC plate. With this year being the fifth year of research on this project, the organic synthesis of HSC has proven to be very difficult and time‐consuming. New approaches have been considered to make the mechanism more efficient. A biochemical pathway of synthesis will now be investigated. High pressure liquid chromatography will be used to explore the separation conditions of materials between steps. The trimerized and macrocyclized version of HSC, known as DFO‐E, is $400/mg if purchased commercially, and HSC is not available at all. If DesD, a potential new antibiotic drug target, is to be fully investigated, efficient, and cost‐effective, means of creating HSC will be required.Support or Funding InformationThis research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.