Weakly interacting cyclodextrin complexes: an experimental and theoretical methodological assessment using two fluorescent anilinonaphthalenes

Abstract 2-Anilinonaphthalene-6-sulfonate (2,6-ANS) and 8-anilinonaphthalene-1-sulfonate (8,1-ANS) are polarity-sensitive fluorescent probes that serve as model systems for host–guest interactions with cyclodextrins. Reliable binding free energies with $$\alpha$$ -, $$\beta$$ -, and $$\gamma$$ -cyclodextrins are needed to exploit these probes as inexpensive, high-throughput reporters for inclusion complexes. Here, we determine the free energies of association of 2,6-ANS and 8,1-ANS to the three native cyclodextrins using fluorescence spectroscopy, isothermal titration calorimetry (ITC), and molecular simulations from either docking or MM/GBSA. Fluorescence titrations indicate that 2,6-ANS binds most strongly to $$\beta$$ -CD, whereas 8,1-ANS binds most strongly to $$\gamma$$ -CD, while ITC places the 8,1-ANS $$\gamma$$ -CD interaction among the weakest of the complexes studied. Docking simulations qualitatively support the experimental trends, whereas MM/GBSA exhibits systematic deviations, particularly for $$\gamma$$ -CD, consistent with limitations of the surface-area-based nonpolar solvation model and parameterization for carbohydrate hosts. Method disagreements are most pronounced for systems with low association constants ( $$5 \times 10^{1}\ \textrm{M}^{-1}$$ ), underscoring the need for cautious interpretation in this weak-binding regime. Molecular dynamics simulations further show that the poor fit of the naphthalene ring in the 2,6-ANS $$\alpha$$ -CD complex can lead to partial solvent exposure and distorted fluorescence readouts. For 8,1-ANS, its larger size amplifies methodological differences, with $$\gamma$$ -CD being the only host, among those examined, that can fully accommodate the naphthalene group within its cavity. Overall, these results illustrate both the utility and the limitations of combining experimental and computational approaches to analyze weak cyclodextrin inclusion complexes.