Design and Synthesis of a Novel in Chemico Reactivity Probe N,N-dimethyl N-(2-(1-naphthyl)acetyl)-l-cysteine (NNDNAC) for Rapid Skin Sensitization Assessment of Cosmetic Ingredients

Skin sensitization is a critical endpoint in cosmetic safety assessment, necessitating reliable animal-free testing alternatives. Current established in chemico assays, such as the Direct Peptide Reactivity Assay and Amino acid Derivative Reactivity Assay, are limited by prolonged 24 h incubation periods and their inability to distinguish between direct electrophilic sensitizers and pro-electrophiles requiring metabolic activation or spontaneous oxidation. This study presents the design, synthesis, and validation of NNDNAC (N,N-dimethyl N-(2-(1-naphthyl)acetyl)-l-cysteine), a novel nucleophilic reactivity probe synthesized via a seven-step pathway. A modified naphthalene structure featuring N,N-dimethylamino substituent enhances nucleophilicity of the cysteine sulfur atom, enabling rapid reactivity assessment within an hour incubation using LC-DAD quantification. Comparative validation studies demonstrated that NNDNAC rapidly identified strong electrophilic sensitizers, achieving 100% and 98% depletion rates for p-benzoquinone and 2-methyl-4-isothiazolin-3-one, respectively, within 1 h. Critically, the NNDNAC assay successfully differentiated pro-electrophiles like p-phenylenediamine and 4-aminophenol, which showed negligible depletion at 1 h but significant depletion after 24 h due to auto-oxidation. Furthermore, NNDNAC classified farnesal as a weak sensitizer, aligning with established KeratinoSens™ and LLNA data. The NNDNAC probe represents a significant advancement in skin sensitization assessment, offering a time-efficient, high-throughput platform that not only accelerates screening processes but also provides crucial mechanistic insights through electrophile/pro-electrophile differentiation, significantly improving animal-free toxicological evaluations.