Oxidation of Free L-histidine by tert-Butylhydroperoxide

• Published in: Pharmaceutical research 2010-03, Vol.27 (3), p.447-456
• Main Authors: Mason, Bruce D, McCracken, Melissa, Bures, Edward J, Kerwin, Bruce A
• Format: Article
• Language: English
• Subjects: Amino acids; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Catalytic oxidation; Cations - chemistry; Cellular biology; Chemical compounds; General pharmacology; Histidine - chemistry; Medical Law; Medical sciences; Metals - chemistry; Oligopeptides - chemistry; Oxidation-Reduction; Pharmaceutical sciences; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Pharmacology/Toxicology; Pharmacy; Proteins; Research Paper; tert-Butylhydroperoxide - chemistry
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-009-0032-y

Purpose L-histidine, a commonly used buffer for protein formulations, has the potential to oxidize and form multiple byproducts. Previous studies were performed using metal catalyzed oxidation with Fe²⁺ or Cu²⁺. We re-examined the oxidation of L-histidine under conditions more appropriate to protein formulations. Methods Solutions of free L-histidine, protected from light, were initially reacted with tert-butylhydroperoxide and the products analyzed by UV absorption spectroscopy, reversed phase HPLC and mass spectrometric analysis and NMR. Experimental parameters investigated were oxidizing agent, pH, temperature, metal ion and metal chelator content. Results The initial reaction produced a number of known products, along with an unknown product that was identified as 4(5)-imidazolecarboxaldehyde. The reaction was highly pH and oxidizing-agent specific. The product was not observed at pH 5.0 or below, while there was a dramatic increase for reactions carried out at pH 6.0 or above. Addition of FeSO₄ to the reaction dramatically increased the amount of 4(5)-imidazolecarboxaldehyde produced, while addition of the metal chelators EDTA or DTPA completely inhibited the reaction. Conclusions The presence of oxidants and trace concentrations of metal ions in high purity L-histidine solutions results in the formation of 4(5)-imidazolecarboxaldehyde which has the potential to covalently modify proteins.