Article pubs.acs.org/JPCA
Excited-State Dynamics of Bis-dehydroxycurcumin Carboxylic Acid, a Water-Soluble Derivative of the Photosensitizer Curcumin Luca Nardo,*,† Angelo Maspero,†,∥ Marco Selva,† Maria Bondani,‡,⊥ Giovanni Palmisano,†,∥ Erika Ferrari,§,# and Monica Saladini§,# †
Department of Science and High Technology, University of Insubria, Via Valleggio 11, 22100 Como, Italy Institute for Photonics and Nanotechnologies, C.N.R., Via Valleggio 11, 22100 Como, Italy § Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via Campi 183, 41125 Modena, Italy ‡
ABSTRACT: Bis-dehydroxycurcumin carboxylic acid (K2A23) is a synthetic curcuminoid designed to exhibit enhanced water solubility and photosensitizing potential with respect to natural curcumin. In this work, the tendency of the compound to form intra- and intermolecular hydrogen bonds in the ground state is studied by UV−visible absorption and by nuclear magnetic resonance (NMR). The excited-state dynamics of the drug are probed in different environments by means of time-correlated single-photon counting measurements and related to its hydrogen bonding affinity in the excited state.
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INTRODUCTION Curcumin (CURC), the dietary pigment extracted from turmeric, is considered one of the most promising natural drug substances.1 Indeed, besides its historically certified antiinflammatory2 and antioxidant3 activity, during the last two decades the compound has been the subject of extensive studies that have demonstrated its efficacy in the treatment of severe diseases such as Alzheimer's,4 cystic fibrosis,5 and AIDS.6 Another particularly interesting feature of CURC as a tentative pharmacological active principle resides in its notable chemopreventive,7 chemotherapeutic,8 and antiproliferative potentials.9 More recently, the phototoxic potential of CURC was also recognized.10−13 From a chemical standpoint, as shown in Figure 1a, CURC belongs to the group of β-diketones. These compounds exhibit tautomerism between several enol and keto structures, which are shown in Figure 2a,b, respectively.14 The closed cis-enol structures are characterized by an intramolecular H-bond between the keto oxygen and the enol proton (keto−enol H-bond), whose formation confers enhanced thermodynamic stability to these conformers. In the early 1990s Gilli et al.15,16 demonstrated that, for a number of simple β-diketones, the strength of the keto−enol H-bond (quantified in terms of the O---O and O−H distances) was strongly correlated to the π-system delocalization. Such a correlation was qualitatively interpreted in terms of a synergistic interplay between double-bond resonance and keto−enol Hbond formation, which the Authors called resonance-assisted H-bonding. In the resonance-assisted H-bonding model, the residual electronic charges on the two oxygen atoms of the β© 2012 American Chemical Society
Figure 1. Molecular structures of (a) curcumin and (b) K2A23.
diketo fragment are maintained close to zero by a feedback mechanism combining the residual charge increase experienced by one oxygen due to resonance and the corresponding charge decrease on the same oxygen due to proton shift. It is quite intuitive, as promptly pointed out by the Authors, that the resonance-assisted H-bonding process is notably inhibited by the presence of carbonyl substituents. In CURC, which is a noncarbonyl-substituted β-diketone, the keto−enol H-bond is sufficiently strong to bias the keto−enol equilibrium. Namely, CURC in solution at room temperature is virtually present in its enol conformers only. The H-bonded closed cis-enol Received: August 9, 2012 Revised: August 30, 2012 Published: August 30, 2012 9321
dx.doi.org/10.1021/jp307928a | J. Phys. Chem. A 2012, 116, 9321−9330
The Journal of Physical Chemistry A
Article
bonding model.15,16 Consequently, a keto−enol equilibrium significantly shifted toward the diketo structures with respect to that displayed by CURC is expected in solution. Interestingly, ab initio calculations showed that the cis-diketo conformer of the curcuminoid structure is much more polar than any of the enol structures.17 Moreover, the diketo moiety can accept two H-bonds. Finally, the pKa of the mobile proton is much higher when the latter is bound to the carbonyl than when it is bound to one of the keto oxygens. Consequently, hydrolytic dissociation is slow even at alkaline pH. These features, together with deprotonation of the carboxyl moiety in neutral and alkali aqueous environments, make the cis-diketo conformer relatively hydrophilic.33 Concerning the issue of excitedstate stabilization, S1 deactivation by means of direct ESIPT can obviously not take place in any of the diketo structures. Moreover, K2A23 lacks the hydroxyl phenolic substituents that are involved in the IMET mechanisms, leading to the fast decay of CURC in an H-bonding environment.30,32 In summary, besides the ascertained enhanced water solubility, K2A23 should also display improved excited-state stability with respect to CURC. In this work, the equilibrium between the enol and diketo conformers of K2A23 is probed in several organic solvents differing in polarity and H-bond formation affinity by means of UV−visible absorption spectroscopy. Considerations upon the strength of the keto−enol H-bond are derived from the relative abundance of diketo conformers. For a selection of solvents, nuclear magnetic resonance studies are also presented which, besides corroborating the conclusions driven from the electronic absorption spectroscopy data, allow unravelling the pattern of intra- and inter- molecular H-bonds formed by K2A23 in the ground state. Finally, the stability of the K2A23 S1-state is determined by time-correlated single-photon counting (TCSPC) measurements and compared to that of CURC. The observed excited-state dynamics is interpreted by assigning a decay mechanism to each of the exponential components resolved in the fluorescence decays. The role of Hbonding in triggering the S1 deactivation is examined.
Figure 2. (a) Enol and (b) diketo conformers of a β-diketone.
structure is dominant in the gas phase as well as in nonpolar environments.17−20 However, in polar environments the keto− enol H-bond is efficiently perturbed and the open cis- and transenol conformers are dominant, respectively, in weakly- and strongly-H-bonding solvents.19 Tiny amounts of the minimally polar trans(anti)-diketo conformer can be found only in nonpolar environments.17 The maximally polar cis-diketo conformer was reported to be not stable for CURC due to the combination of unfavorable dipole−dipole alignment and steric interactions.17,20 The main obstacle toward exploitation of CURC as a pharmaceutical active principle resides in its poor water solubility, which in turn results in scarce bioavailability of the compound.21 Curcumin is practically insoluble in water at acidic or neutral pH.22 Although the compound is soluble in alkali, as soon as dissociation of the enolic proton takes place (pKa ≈ 8.5) the compound undergoes a rapid hydrolytic degradation to feruloyl methane and ferulic acid.23 The notable instability of the CURC excited state is an additional issue in view of its formulation into a photosensitizer. In a previous work,24 we proposed a model which was capable of explaining the data on the decay from the S1-state of CURC. The following radiationless decay mechanisms were considered to concur with fluorescence emission: (i) direct excited-state intramolecular proton transfer (ESIPT) from the enol to the keto group of the closed cis-enol tautomer;25 (ii) slow, solventrearrangement moderated ESIPT. The latter occurs in case a trans-enol or open cis-enol molecule isomerizes to the closed cis-enol conformer whereas in the S1-state, and then decays to S0 by means of ESIPT;24 (iii) reketonization (excited-state transfer of the enolic proton to the carbonyl moiety);25,26 (iv) intermolecular charge/energy transfer (IMET) to the solvent molecules.27 We showed that the fastest nonradiative S1-decay process for CURC in an inert environment is direct ESIPT, whereas in strongly-H-bonding solvents, where the keto−enol H-bond is disrupted, IMET is the main decay mechanism. In a series of subsequent articles,28−32 we analyzed curcuminoids differently substituted at the phenyl rings and observed that IMET occurs only if both the methoxy substituents in the meta-position and the hydroxyl substituents in para-position of the parent compound are present. With the aim of developing the curcuminoid structure into a photosensitized drug substance capable of overcoming the issues plaguing CURC, we recently synthesized the compound K2A23 (Figure 1b), a carboxylic acid of the phenyl substituted curcuminoid bis-dehydroxycurcumin.33 In K2A23, the presence of the carboxylic moiety perturbs the resonance of the doublebonds in the keto−enol ring; thus it is expected to weaken the keto−enol H-bond according to the resonance-assisted H-
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MATERIALS AND METHODS Chemicals and Sample Preparation. K2A23 was synthesized as previously described.33 Solutions for UV−vis absorption spectroscopy, steady-state fluorescence measurements, and time-resolved fluorescence studies were prepared in the solvents listed in Table 1. In the same table, viscosity, polarity (expressed in terms of the dielectric constant, ε), and Table 1. Chemical-Physical Properties of Selected Solvents solvent nonpolar polar weakly-Hbonding
H-bond acceptors alcohols
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cyclohexane chloroform ethyl acetate acetone acetonitrile DMF DMSO 2-propanol ethanol methanol ethylene glycol
η (cP)
ε
α
β
0.98 0.58
2.02 4.81
0 0.44
0 0
0.45 0.32 0.36 0.90 1.99 2.5 1.2 0.55 16.1
6.02 20.60 38.8 37.6 48.9 19.92 25.07 33.62 37.70
0 0.08 0.19 0 0 0.78 0.83 0.93 0.90
0.45 0.48 0.31 0.69 0.76 0.95 0.77 0.62 0.52
dx.doi.org/10.1021/jp307928a | J. Phys. Chem. A 2012, 116, 9321−9330
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H-bond donating and accepting properties (quantified by means of the Kamlett acidity parameter α and basicity parameter β, respectively34) of the solvents are also reported. All the solvents were ≥99.5% pure and were used as received, except ethyl acetate, which was dried over sodium sulfate. All samples were prepared the same day they were used for measurements. The standard 1H and 13C spectra in solution were carried out in dimethyl sulfoxide (DMSO-d6), chloroform (CDCl3), and acetonitrile (MeCN-d3), at room temperature. Steady-State Spectroscopy. The UV−vis absorption spectra were measured by a Perkin-Elmer Lambda 2 UV−vis spectrophotometer. The fluorescence emission and excitation spectra were acquired with a PTI Fluorescence Master System spectrofluorometer. The acquisition software provided online correction for both the excitation lamp and the detector spectral responses. Fluorescence quantum yields were determined by comparison with a solution of dimethyl-popop in ethanol (ΦFl = 0.95), excited at the 363 nm absorption peak, by normalizing with respect to the relative absorption and solvent refractive index. NMR measurements were performed with a Bruker ADVANCE 400 spectrometer. Time-Resolved Fluorescence. Time-resolved fluorescence decay distributions were reconstructed by means of a TCSPC apparatus endowed with
