Drug–protein interactions assessed by fluorescence measurements in the real complexes and in model dyads Ignacio Vayá, Raúl Pérez-Ruiz, Virginie Lhiaubet-Vallet, M. Consuelo Jiménez * , Miguel A. Miranda * Departamento de Quimica/Instituto de Tecnología Química, UPV-CSIC, Universidad Politécnica de Valencia, Camino de vera s/n, 46071, Valencia, Spain article info Article history: Received 26 October 2009 In final form 29 December 2009 Available online 13 January 2010 abstract In the present work, a systematic fluorescence study on supramolecular systems using two serum albu- mins (HSA or BSA) as hosts and the nonsteroidal antiinflammatory drugs carprofen (CPF) or naproxen (NPX) as guests has been undertaken. In parallel, model dyads containing Tyr or Trp covalently linked to CPF or NPX have also been investigated. In HSA/(S)-CPF and BSA/(S)-CPF (k exc = 266 nm), at 1:1 M ratio, an important degree (more than 40%) of singlet–singlet energy transfer (SSET) was observed to take place. The distance (r) calculated for energy transfer from the SAs to (S)-CPF through a FRET mechanism was found to be ca. 21 Å. In the case of HSA/(S)-NPX and BSA/(S)-NPX, energy transfer occurred to a lower extent (ca. 7%), and r was determined as ca. 24 Å. In order to investigate the possible excited state inter- actions between bound ligands and the relevant amino acids present in the protein binding sites, four pairs of model dyads were designed and synthesised, namely (S, S)-TyrCPF, (S, R)-TyrCPF, (S, S)-TrpCPF, (S, R)-TrpCPF, (S, S)-TyrNPX, (S, R)-TyrNPX, (S, S)-TrpNPX and (S, R)-TrpNPX. A complete SSET was observed from Tyr or Trp to CPF, since no contribution from the amino acids was present in the emission of the dyads. Likewise, a very efficient Tyr or Trp to NPX energy transfer was observed. Remarkably, in (S, S)-TrpNPX and (S, R)-TrpNPX a configuration-dependent reduction in the emission intensity was observed, revealing a strong and stereoselective intramolecular quenching. This effect can be attributed to exciplex formation and is dynamic in nature, as the fluorescence lifetimes were much shorter in (S, R)- and (S, S)-TrpNPX (1.5 and 3.1 ns, respectively) than in (S)-NPX (11 ns). Ó 2009 Elsevier B.V. All rights reserved. 1. Introduction Incorporation of drugs containing an active chromophore into more complex structures can give rise to relevant intramolecular excited state interactions with a number of covalently linked sub- structures. This has been used for the design of a wide variety of model systems aimed at providing mechanistic insight into the fundamental aspects of key processes such as energy transfer, elec- tron transfer or exciplex formation [1–18]. Photoinduced energy transfer between a donor and an acceptor can occur via Coulombic interaction (Förster mechanism) and/or electronic exchange interaction (Dexter mechanism) [19,20]. The former operates through space and does not require orbital overlap of donor and acceptor. It is effective when the involved transitions have high oscillator strengths, typically in the case of spin allowed processes such as singlet–singlet energy transfer. This mechanism can occur over distances up to 100 Å. Förster resonance energy transfer (FRET) has been extensively used in a number of fields [21–31], including biochemistry or medical diagnostic, since the favourable distances for FRET are comparable to the size of a protein or the thickness of a membrane. In medicinal chemistry, fluorescence emission has shown to be a very useful tool to gain in- sight into the interactions that take place between drugs and trans- port proteins. An interesting feature of proteins is that, by contrast with other biomolecules, they display useful intrinsic fluorescence due to the presence of amino acids such as phenylalanine (Phe), tyrosine (Tyr) and/or tryptophan (Trp) [32]. In general, their fluorescence spectrum is dominated by Trp, which is highly sensitive to local environment; therefore, ligand binding to proteins can result in changes of Trp emission. In this context, energy transfer can be used to determine the distance from a Trp residue to a complexed drug. Serum albumins (SAs) are the most abundant soluble proteins in plasma of humans and other mammalians. They are responsible for a variety of fundamental functions, including transport of endogenous and exogenous agents, maintenance of pH and colloid osmotic pressure of plasma and extra vascular fluids, lipid metab- olism etc. [33,34]. Human serum albumin (HSA) has a typical con- centration of 5 g/100 mL in the circulatory system. Its structure consists of 585 amino acids, and its molecular mass is 65 kDa. The presence of a considerable amount of cysteine (6%), mostly in- volved in disulphide bridges, provides stability to the HSA struc- ture. In addition, there are a large number of charged amino acids (31%) and only one Trp residue that has been particularly 0009-2614/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2009.12.091 * Corresponding authors. Fax: +34 963877809. E-mail addresses: mcjimene@qim.upv.es (M.C. Jiménez), mmiranda@qim.upv.es (M.A. Miranda). Chemical Physics Letters 486 (2010) 147–153 Contents lists available at ScienceDirect Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett