p-Nitrophenoxy analogs as practical surrogates for Novichok-related biomarker verification in forensic toxicology

Authentic Novichok (A-series) nerve agents are highly restricted, making the rapid preparation of positive controls and reference materials challenging in forensic toxicology. This study aimed to evaluate p-nitrophenoxy analogs as surrogate compounds for Novichok-related biomarker verification by assessing their synthetic accessibility, ability to generate diagnostically relevant butyrylcholinesterase (BuChE) nonapeptide biomarkers, and functional relevance in cholinesterase inhibition and reactivation assays.

Six surrogate compounds representing phosphate- and phosphonate-type analogs were prepared using a revised synthetic sequence with scaffold-dependent optimization of the reaction and purification conditions. Their utility was evaluated using plasma BuChE inhibition assays, 2-pyridine aldoxime methochloride (2-PAM)-mediated reactivation studies, inhibition kinetics with horse BuChE and human acetylcholinesterase, and liquid chromatography-tandem mass spectrometry analysis of BuChE-derived nonapeptide adducts in plasma. Operational applicability was examined using the OPCW biomedical proficiency test (BioPT) evaluation criteria.

All six surrogate compounds were obtained by structural class-dependent optimizations of their synthesis and purification. The plasma BuChE IC₅₀ values of authentic A230, A232, A234, A242, A262, and A2xx were 12.3, 9.6, 8.6, 10.1, 10.6, and 24.5 ng/mL, respectively, whereas those of the corresponding surrogates were 58.5, 38.6, 55.2, 3942, 3829, and 2575 ng/mL. 2-PAM-mediated BuChE reactivation showed similar qualitative patterns within each authentic/surrogate pair. All surrogates generated analytically equivalent BuChE nonapeptides and could be used to prepare qualitative reference materials for the OPCW BioPT-related workflow.

p-Nitrophenoxy Novichok-related analogs provide a practical surrogate platform for preparing diagnostically relevant BuChE nonapeptide reference materials without using authentic A-series nerve agents.