N-hydroxysuccinimidyl derivatization reagents, friends or foes? From synthesis to derivatization of amino compounds and their analysis using liquid chromatography tandem mass spectrometry

Abstract N-Hydroxysuccinimidyl (NHS) esters are widely employed in peptide coupling; however, their behavior in derivatization for high-performance liquid chromatography (HPLC) analysis differs from the synthesis point of view. This work evaluated the performance of NHS derivatization reagents for amino compounds using liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI–MS/MS); later, their E/Z photoisomerization was studied. These reagents were azobenzene-based carbamates (AzoA and AzoB), or esters (AzoC and AzoD), fluorescent NHS-esters incorporating Coumarin and Pyrene moieties. It was observed that NHS-esters are susceptible to different side reactions (alkaline hydrolysis, alcoholysis, and transesterification), which later affect their performance, especially if the goal is to analyze β-Alanine (β-Ala). To highlight this potential pitfall for analytical chemists, the extent of these side reactions was studied in different conditions: e.g., the base used, aqueous borate buffer or tertiary amines if the reaction is carried out at room temperature or 50 °C, and with or without alcohol. It was found that, in contrast to NHS-esters, NHS-carbamates are free from β-Ala interfering (confirmed with first-time synthesized carbamate AzoA). Furthermore, the study explored replacing the NHS group in AzoD ester (commercially known as Dabcyl-OSu, Dabcyl-SE or Dabcyl-NHS) with 1-hydroxybenzotriazolyl leaving group, creating AzoD-OBt. The LC–ESI–MS/MS study shows that the AzoD-OBt reagent can overcome the side reaction of NHS-ester, which would benefit the derivatization yield toward the analytes.