Two designed haptens for fluorene: Synthesis, crystal structure, Hirshfeld surface, quantum chemical studies, and potential for conjugation with carrier proteins

Polycyclic aromatic hydrocarbons (PAHs) are considered major environmental pollutants and are known to pose serious threats to both ecosystems and human health. Immunoassay techniques have been identified as highly promising tools for the rapid detection of PAHs. These techniques rely on the specific antigen–antibody recognition, where the selectivity and sensitivity of the assay are primarily determined by antibody performance, which is, in turn, influenced by the design and properties of the hapten and artificial antigen. In this work, two fluorene-specific haptens were synthesized, (Hapten 1: 4-(9H-fluoren-2-yl)-4-oxobutanoic acid, and Hapten 2: 6-(9H-fluoren-2-yl)-6-oxohexanoic acid), and their structures were characterized using single-crystal X-ray diffraction. The stability of hapten 1′s structure is conferred by O–H⋅⋅⋅O hydrogen bonding and weak intermolecular forces. The stability of hapten 2′s structure is achieved through O-H···O and C-H···O hydrogen bonding, along with weak intermolecular forces. Hirshfeld surface analysis quantified the patterns of intermolecular interactions. DFT calculations at the B3LYP/6-31++G(d,p) level analyzed the frontier orbitals, ADCH charges, molecular electrostatic potentials, and natural bond orbitals of the haptens. The HOMO-LUMO gaps of haptens 1 and 2 were calculated as 4.278 eV and 4.311 eV, respectively. These HOMO-LUMO gaps suggest the target molecules' appropriate softness and reactivity. Molecular docking studies showed high binding affinity between the target haptens and carrier proteins. Hapten 1 demonstrated a docking score of -7.56 kcal/mol with BSA, and -7.04 kcal/mol with OVA. Hapten 2 demonstrated a docking score of -8.37 kcal/mol with BSA, and -6.56 kcal/mol with KLH. The results of molecular dynamics simulation show that the BSA-Hapten 1, BSA-Hapten 2, OVA-Hapten 1, and KLH-Hapten 2 complex systems are stably bound and the complexes have good hydrogen bonding. Immunogens were successfully prepared by conjugating haptens 1 and 2 with BSA, achieving coupling rates of 16.1 and 13.8, respectively. Hapten 1 was conjugated to OVA with a conjugation ratio of 11.4. Hapten 2 was conjugated with KLH, preparing the coated antigen. This research delivers comprehensively characterized fluorene haptens, establishing a robust foundation for the future development of sensitive and specific immunodetection methods targeting fluorene.