Caging and solvent effects on the tautomeric equilibrium of 3-pyridone/3-hydroxypyridine in the ground state: a study in cyclodextrins and binary solvents Osama K. Abou-Zied* and Othman I. K. Al-Shihi Received 7th January 2009, Accepted 25th March 2009 First published as an Advance Article on the web 20th April 2009 DOI: 10.1039/b823405a The tautomeric equilibrium between 3-pyridone (3Py) and 3-hydroxypyridine (3HP) shows characteristic absorption peaks for the zwitterion form of 3Py in water that may be used as a probe of the hydrophobic nature inside macromolecules such as proteins and other biologically related systems. We studied this equilibrium in the ground state in aqueous cyclodextrins (CDs) and in binary solvent mixtures of 1,4-dioxane and water by absorption spectroscopy, and by ab initio calculations. Upon the addition of a-CD or b-CD to an aqueous solution of the 3Py/3HP system, the absorbance intensity of the zwitterion tautomer decreases with a concomitant increase in the intensity of the enol tautomer of 3HP. The results reflect the nature of the tautomeric equilibrium and point to the hydrophobic environment inside the CD cavities. The effect of inclusion is noticeably less in the case of a-CD. This is attributed to the small cavity size of a-CD which sustains only partial inclusion. Upon the addition of g-CD, the intensity of the zwitterion tautomer slightly increased over that in water which is attributed to the direct interaction between the charged sides of the tautomer with the outer primary or secondary hydroxyls of the glycopyranose units of g-CD. This interaction is a result of the large cavity size of g-CD which does not support a stable complex. The largest caging effect was observed in 2,6-di-O-methyl-b-CD (DMb-CD) which is an indication of a more hydrophobic environment around the guest. The large hydrophobicity of DMb-CD is due to the presence of the two methyl groups in the b-CD derivative which reduce the amount of water inside the cavity upon encapsulation. In the binary mixtures of 1,4-dioxane and water, the change in the absorbance intensity of the enol and the zwitterion tautomers was analyzed quantitatively and three water molecules were found to solvate the polar centers of each tautomer. Ab initio calculations of the solvation of both tautomers by two and three water molecules were performed at the MP2/6-31++G(d,p) level. The calculations show that three water molecules are necessary to solvate the polar centers of each tautomer in a water network pattern. The results presented here suggest that the 3Py/3HP system represents a potentially useful new photophysical probe for supramolecular structures, particularly those involving inclusion. 1. Introduction The interior properties of a biological macromolecule are crucial to its structure and functionality. A large number of low molecular weight compounds bind reversibly to proteins and are widely used as extrinsic probes for the investigation of physicochemical, biochemical and biological systems. The spectral changes observed on the binding of probes with proteins are an important tool for the investigation of binding sites, conformational changes and characterization of substrate to ligand binding. 1 Recently, we used three isomers of small aromatic molecules as probes to characterize the drug-binding site ‘‘subdomain IIA’’ in human serum albumin (HSA). 2 2-Pyridone (2Py), 3-pyridone (3Py) and 4-pyridone (4Py) (shown in Scheme 1) have received much interest due to the similarity of their molecular structures with those found in a wide range of drugs of different pharmacological functions. 3 The three probes have the same functional groups and similar chemical properties, which include the possibility of keto-enolic tautomerism (solvent polarity dependent). 4–12 We found that the probes specifically bind in subdomain IIA and cause a reduction in the fluorescence intensity and lifetime of the Trp-214 residue in native HSA which is located in the same subdomain. The efficiency of energy transfer from the Trp-214 fluorescence to the probes was analyzed using Fo¨rster theory and was found to depend on the degree of the spectral overlap between the donor’s fluorescence and the acceptor’s absorption. Studies of the above pyridone systems in different solvents proved that the 2- and the 4-isomers are distinctly different from the 3-isomer. 5–8 The former isomers exist predominantly in the pyridone form in neutral solution, whereas evidence for a zwitterion has been reported for the 3-isomer. 11,12 While in Department of Chemistry, Faculty of Science, Sultan Qaboos University, P.O. Box 36, Muscat, Postal Code 123, Sultanate of Oman. E-mail: abouzied@squ.edu.om; Fax: +968 2414 1469; Tel: +968 2414 1468 This journal is  c the Owner Societies 2009 Phys. Chem. Chem. Phys., 2009, 11, 5377–5383 | 5377 PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics