Detection of 1-Hydroxypyrene as biomarker for polycyclic aromatic hydrocarbons via cyclodextrin-based systems

1-Hydroxypyrene (1HOPy) is the major urinary metabolite of pyrene and one of the most widely used biomarkers for assessing human exposure to polycyclic aromatic hydrocarbons (PAHs). Despite its analytical relevance, the molecular determinants governing the interaction of 1HOPy with supramolecular hosts in aqueous solution remain poorly understood. In this work, we present a combined spectroscopic and computational investigation of the binding of 1HOPy to β-cyclodextrin (βCD), γCD (γCD), hydroxypropyl-βCD (HPβCD), dimers of βCD and two polymers with the aim of elucidating the structural and thermodynamic factors underlying complex formation. Steady-state absorption and fluorescence spectroscopy, and time-resolved fluorescence measurements reveal distinct binding behaviours depending on the host architecture. While βCD, HPβCD and the dimers predominantly form 1:1 complexes with 1HOPy, γCD displays cooperative binding, giving rise to both 1:1 and 2:2 host–guest assemblies. In the polymers 2 CDs tend to encapsulate the 1HOPy guest. Spectroscopic signatures further indicate that, under buffered conditions, partial formation of the enolate form of 1HOPy may contribute to enhanced stabilization in one dimer. Binding constants differ even by 2 orders of magnitude with HPβCD displaying the highest affinity followed by one dimer. To rationalize these observations at the molecular level, extensive molecular dynamics simulations combined with thermodynamic integration (TI) were performed. Computational models were designed to reflect the experimentally relevant stoichiometries while also probing alternative binding scenarios to explain deviations from experimental fits. Structural analyses based on RMSD, minimum host–guest distances, hydrogen-bond patterns, and clustering reveal that βCD dimers remain relatively flexible and poorly compacted upon guest binding, whereas γCD undergoes pronounced guest-induced dimer stabilization. In particular, inclusion of two 1HOPy molecules drives the compaction of the otherwise labile γCD dimer, leading to a rigid and cooperative supramolecular assembly. Binding free energies computed by TI reproduce the experimental hierarchy of affinities and highlight that interaction energies alone do not predict thermodynamic stability. For βCD systems, strong local host–guest interactions translate only into moderate binding free energies, whereas γCD exhibits a marked amplification of affinity arising from cooperative host–host reorganization. Comparison between calculated and experimental free energies shows overall agreement, while also delineating specific deviations attributable to chemical effects not explicitly captured by the classical force-field description. Overall, this study demonstrates that 1HOPy not only acts as a passive guest but can actively stabilize supramolecular architectures, particularly in γCD systems. These findings provide molecular-level insight into cyclodextrin–PAH metabolite recognition and support the rational design of cyclodextrin-based platforms for the detection and sensing of environmental biomarkers.