Method of synthesis and purification of citrulline
Inventors
BULL, James Alan • FISCHER, Holger Christian • KAISER, Karl Juergen • MONNOT-CHASE, Bette
Assignees
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Publication Number
US-12240801-B2
Publication Date
2025-03-04
Expiration Date
Abstract
This invention provides for synthesis of citrulline from a transition metal complex of ornithine using cyanate to derivatize the terminal amino group of ornithine. The invention also provides improved methods for purification of citrulline produced by reaction of cyanate with ornithine via the steps of reprecipitation of copper complex of citrulline, removal of the complexing metal by sulfide precipitation, activated carbon adsorption and antisolvent crystallization.
Core Innovation
The invention relates to an improved citrulline manufacturing process that adds a terminal carbamido group to an alpha amino acid. The method obtains an alpha-amino acid having a terminal amine complexed with copper in aqueous solution, and then reacts the terminal amine with cyanate to form a copper complexed carbamido derivative.
In the described process, reacting the alpha amino acid terminal amine with cyanate results in a copper-bound citrulline complex that precipitates. The precipitate can be reprecipitated to reduce ornithine contamination, including acid dissolve followed by base reprecipitation of the citrulline:copper complex under controlled temperature conditions.
The process further recovers citrulline by removing copper, including precipitating copper as copper sulfide using hydrogen sulfide while maintaining citrulline in solution. Additional purification is directed toward pharmaceutical grade material, including neutralization, activated carbon adsorption, optional nano-filtration/micro-filtration/sterile filtration, and solvent/anti-solvent crystallization with subsequent drying.
The document also states suitability for injectable therapeutic use and references therapeutic indications. Microbial control practices are discussed in the context of manufacturing, including endotoxin control and use of sterile bag and filter press hardware, together with cleanroom/GMP environment considerations.
Claims Coverage
The independent claims are directed to a method for adding a terminal carbamido group to an alpha amino acid by forming an aqueous copper complex of the alpha-amino acid terminal amine, reacting with cyanate, and recovering a copper-complexed carbamido derivative as a precipitate. The dependent claims add specific alpha-amino acid and carbamido derivative pairs, constrain reaction conditions for the cyanate step, and add recovery conditioning such as cooling and washing.
Copper complex in aqueous solution of alpha-amino acid terminal amine
Obtaining an alpha-amino acid having a terminal amine complexed with copper in aqueous solution.
Cyanate reaction of copper-complexed terminal amine
Reacting the alpha amino acid having a terminal amine with cyanate.
Recovering copper-complexed carbamido derivative precipitate
Recovering a carbamido derivative of the alpha-amino acid complexed to copper as a precipitate.
Specific alpha-amino acid and carbamido derivative pairs
The alpha-amino acid with the terminal amine is ornithine and/or lysine, and the carbamido derivative is citrulline and/or homocitrulline.
Temperature and time ranges for the cyanate reaction step
Performing the cyanate reacting step (b) at a temperature between 30°C and 100°C and/or with a reaction time between 0.5 and 5 hours.
Cooling and water washing endpoint for recovered precipitate
Cooling the reaction mixture to at least ambient temperature or lower before recovery, and washing the recovered precipitate with water until no blue color appears.
Across the independent claim and its refinements, the inventive aspects are centered on forming an aqueous copper complex of an alpha-amino acid terminal amine, reacting it with cyanate to generate a copper-complexed carbamido derivative that precipitates, and recovering the precipitate, with refinements for specific substrate/product pairs, reaction temperature/time ranges, and cooling and water-washing conditions.
Stated Advantages
Improved citrulline manufacturing with reduced ornithine contamination via reprecipitation of the citrulline:copper complex.
Purification directed to pharmaceutical grade material suitable for injectable therapeutic use.
Recovery involving copper removal by hydrogen sulfide precipitation while maintaining citrulline in solution.
Documented Applications
Manufacture of citrulline for injectable therapeutic use, with referenced therapeutic indications.
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