Racemization without deamidation: Effect of racemization conditions on 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids

Abstract The aim of this research study was to provide a more thorough understanding of the underlying mechanism and to broaden the application field of the recently introduced racemization method employing the amino acid derivatization tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) for heat-induced stereoisomerization of common amino acids as well as uniformly isotopically labeled [U-13C15N]-amino acids. The influence of different buffer types such as sodium borate buffer and sodium carbonate buffer as well as their pH and molarity on the racemization and deamidation of amino acids were investigated. It was found that a 0.4 M borate buffer with a pH of 8.0 +/− 0.2 was the most suitable derivatization as well as racemization buffer to ensure degradation free racemization of deamidation prone compounds such as glutamine. Hereby essential was the in-solution pH measurement before and after derivatization with AQC as well as after heat-induced racemization. This strategy provided further insight at which pH an actual racemization event was observed and when an unwanted deamidation of glutamine to glutamic acid occurred. In addition also the influence of the presence of oxygen during racemization was studied. In this context it was possible to determine ideal oxidation and racemization conditions for the production of scalemic mixtures of chiral isotopically labeled methionine AQC-DL-[U-13C15N]-Met as well as its oxidation products, AQC-DL-[U-13C15N]-Met-O and AQC-DL-[U-13C15N]-Met-O2. All stereoselective separations were performed on the zwitterionic Chiralpak ZWIX(+) column combined with HPLC–ESI–QTOF–MS analysis in positive ionization mode.