J. Phys. Chem. B 2007, 111, 423-431
423
Stereodifferentiating Drug-Biomolecule Interactions in the Triplet Excited State: Studies on Supramolecular Carprofen/Protein Systems and on Carprofen-Tryptophan Model Dyads Virginie Lhiaubet-Vallet, Francisco Bosca´ , and Miguel A. Miranda* Instituto de Tecnologı´a Quı´mica UPV-CSIC, UniVersidad Polite´ cnica de Valencia, AVenida de Los Naranjos, s/n, 46022 Valencia, Spain ReceiVed: October 24, 2006
Stereoselective interaction between a chiral nonsteroidal antiinflammatory drug, namely carprofen (CP), and human serum albumin (HSA) was studied, and the results were compared with those obtained with model dyads. In the presence of albumin the same triplet-triplet transition was detected for both CP stereoisomers; however, time-resolved measurements revealed a remarkable stereodifferentiation in the CP/HSA interaction. For each stereoisomer, the decay dynamics evidenced the presence of two components with different lifetimes that can be correlated with complexation of CP to the two possible albumin binding sites (site I and site II). This assignment was confirmed by using ibuprofen, a site II displacer. Thus, the shorter lived components, for which stereodifferentiation was more important (τR/τS ca. 4), were ascribed to the CP triplet state in site I; the lifetime shortening can be attributed to electron-transfer quenching by the only tryptophan (Trp) of the protein. Laser flash photolysis of model dyads containing covalently linked CP and Trp revealed formation of the expected Trp radical cation, providing support for such a mechanism. Moreover, significant stereodifferentiation was observed between the (R)- and (S)-CP-Trp dyads. In the case of CP/HSA complexes, as well as in the model compounds, the stereodifferentiation detected in the decays is in good agreement with that observed in the formation of the only CP photoproduct, resulting from a photodehalogenation process. Moreover, stereodifferentiation was also found to occur for the photobinding of CP to the protein.
Introduction Chiral recognition plays a crucial role in chemistry. The concept has been developed in very different fields from pharmaceutical to agricultural and environmental sciences with applications ranging from drug design to material sciences or nanotechnology. Generally, it concerns ground-state interactions; nevertheless, there is an increasing trend toward the investigation of this phenomenon in the excited states. Thus, photochemistry is becoming an important field of application, as indicated by the significant developments achieved in the last 20 years.1 Photoresolution and photochirogenesis have been well substantiated in the literature; however, direct chiral discrimination in the excited triplet state has only been found in a few cases.2-5 Indeed, photochemical asymmetric synthesis is more difficult to control than its ground state counterpart as it involves shortlived and weakly interacting excited states. Hence, supramolecular chemistry has been used to circumvent this disadvantage, with the introduction of chiral host systems and synthetic assemblies such as cyclodextrins,6 modified zeolites,7 or tuned molecules.8 Natural hosts including serum albumins9,10 and DNA11 have also shown a clear potential to promote stereoselective photoreactions. In this context, biomolecules are attractive entities with wide application possibilities in photochemistry, from the investigation of fundamental mechanisms9,10 to the development of tools for molecular biology12 or photocatalytic antibodies.13 Moreover, the photosensitization of biomolecules has recently attracted * Address correspondence to this author. Phone: +34-96 3877807. Fax: +34-96 38 77 809. E-mail:
[email protected].
considerable attention, as a variety of drugs absorbing UV light are able to induce important photobiological damage. Hence, photophysical and photochemical studies in the presence of biomolecules should give a detailed knowledge of stereodifferentiating processes and appears essential for the design of new chiral therapeutic agents aimed at improving the benefit/ risk ratio by decreasing the photosensitivity side effects. Despite the significance of such studies, the photobiological properties of chiral drugs remain practically unexplored. A weak stereodifferentiation has been observed during nucleoside- and DNAphotosensitization by chiral carprofen and ofloxacin, while no stereoselectivity has been detected in lipid photoperoxidation or photohemolysis of red blood cells by ketoprofen.14 Recently, a significant enantioselectivity has been observed during the intermolecular quenching of the ketoprofen triplet state by DNA building blocks, namely thymidine and 2′-deoxyguanosine.2 In addition, bichromophoric compounds designed to mimic interactions between nonsteroidal anti-inflammatory drugs (NSAID) and lipids,3 proteins,5,15 or DNA16 have revealed a high diastereodifferentiation in the intramolecular quenching of the drug triplet excited state. Nevertheless, it is only very recently that analogous chiral recognition at the intermolecular level, i.e., the possibility of stereodifferentiating interactions between the drug triplet excited-state and biomolecules, has been reported.17,18 Albumins have been widely used as the stationary phase for the chromatographic resolution of racemic mixtures.19 There are also some reports on their use in the attempted photoresolution of racemic ketoprofen and naphthols.10 Besides, it is now established that human serum albumin (HSA) is a model of
10.1021/jp066968k CCC: $37.00 © 2007 American Chemical Society Published on Web 12/22/2006
424 J. Phys. Chem. B, Vol. 111, No. 2, 2007 choice to study the photobinding properties of drugs, since this protein plays a very important role in the pharmacokinetics, pharmacodynamics, and toxicology of most of the xenobiotics. Moreover, albumins have been extensively investigated as model systems for drug-induced photoallergy, an immunological response mediated by the photochemical formation of a covalent bond between a protein and a photosensitizer.20 In a preliminary communication,17 we have studied the interactions between a chiral NSAID, namely carprofen (CP), and HSA by laser flash photolysis, showing that the distribution of the drug between the two protein binding sites can be monitored by the dynamics of its triplet state decay. In addition, the stereodifferentiation observed for triplet lifetimes can be correlated with the stereoselectivity observed in photoproduct (PP) formation and in drug photobinding to the protein. An electron-transfer mechanism between excited carprofen and tryptophan has been proposed to explain the experimental observations. In this work, we wish to report in full the results of a more detailed study that has been undertaken to confirm the assignment of the CP triplet state in site I and site II of HSA. Moreover, model systems have been used to investigate the mechanism of CP photolysis when complexed with HSA, but also that of CP-HSA photobinding. Specifically, in view of the central role of tryptophan in the involved processes, the noncovalent CP/Trp interactions in the real complexes have been modeled through the analogous intramolecular interactions in covalently linked CP-Trp bichromophoric dyads. Indeed, quenching has been found to occur in the model dyads with a rate constant that is markedly configuration dependent. Materials and Methods Chemicals. Human serum albumin (essentially free from fatty acid), guanidinium hydrochloride, phosphate buffer (10 mM in NaCl pH 7.4), (S)-ibuprofen (IB), tryptophan methyl ester, and glycine ethyl ester were purchased from Sigma. G-25 Sephadex (PD10) columns were obtained from Amersham Pharmacia. N’(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and (S)-ketoprofen were from Aldrich and 1-hydroxybenzotriazole (HOBt) solution from Fluka. (R)-Ibuprofen was purchased from Toronto Research Chemicals Inc. Racemic carprofen was Soxhlet extracted with methanol from commercial samples of Rimadyl, produced by Pfizer (Madrid, Spain). Separation of (R)- and (S)-carprofen was performed by high-performance liquid chromatography (HPLC) on a Varian apparatus equipped with a 9012Q pump, a Prostar 335 photodiode array detector, and a chiral phase column (Kromasil CHITBB, 5 µm packing) with hexane:tert-butyl methyl ether:acetic acid (60/40/0.1, v/v) as the mobile phase. All quality HPLC solvents were obtained from Merck. Instrumentation. Photoreactor. UVA irradiations were performed by means of a multilamp photoreactor equipped with 10 lamps (Osram Sylvania, F15T8/BLB) emitting from 310 to 410 nm with a maximal output (1 mW/cm2) at ca. 360 nm. Laser Flash Photolysis. A pulsed Excimer laser system with Xe/HCl/Ne mixture was used for the excitation at 308 nm. The single pulses were ∼17 ns duration and the energy was e100 mJ/pulse. A pulsed Lo255 Oriel xenon lamp was employed as the detecting light source. The laser flash photolysis apparatus consisted of the pulsed laser, the Xe lamp, a 77200 Oriel monochromator, and an Oriel photomultiplier tube (PMT) system made up of a 77348 side-on PMT tube, 70680 PMT housing, and a 70705 PMT power supply. The oscilloscope was
Lhiaubet-Vallet et al. a TDS-640A Tektronix. The output signal from the oscilloscope was transferred to a personal computer. Fluorescence. The steady-state fluorescence experiments were carried out on a Photon Technology International (PTI) LPS220B spectrofluorometer. All the spectra were recorded after excitation at 320 nm. Fluorescence quantum yield measurements were performed with nitrogen-bubbled acetonitrile solutions, the absorbance was adjusted at the excitation wavelength (λ ) 320 nm), and carbazole or carprofen was used as standard (φf ) 0.42 or 0.068, respectively).21 Lifetimes were measured with a Time Master fluorescence lifetime spectrometer TM-2/2003 from PTI by means of the stroboscopic technique, which is a variation of the boxcar technique. A hydrogen/nitrogen flash lamp was used as the excitation source. The kinetic traces were fitted by monoexponential functions, using a deconvolution procedure to separate from the lamp pulse profile. Synthesis of the CP-Trp and CP-Gly Dyads. Racemic CP (100 mg, 3.65 × 10-4 mol), L-Trp methyl ester (108 mg, 4.26 × 10-4 mol), or L-Gly ethyl ester (60 mg, 4.26 × 10-4 mol), EDC (81.7 mg, 4.26 × 10-4 mol), and HOBt (4.26 × 10-4 mol) were combined in dry DMF (10 mL), and the mixture was stirred overnight. Then, the solution was treated with 10% HCl and extracted with ethyl acetate. The organic phase was washed 3 times with water and twice with saturated sodium bicarbonate and then dried over sodium sulfate. The residue was dried under vacuum to give a yellowish solid. In the case of CP-Trp, the two diastereoisomers were separated by semipreparative HPLC (reverse phase column C18 Kromasil 100, 25 × 1.0 cm, acetonitrile:water, 1/1, v/v). The stereochemistry was assigned by comparison with the product of a similar synthesis (in smaller amounts) performed with the pure (R)-CP isomer. Preparation of PP-Trp. Deaerated solutions of (R)-CP-Trp or (S)-CP-Trp in acetonitrile were UVA irradiated, and the photomixtures were separated by semipreparative reverse-phase HPLC (same conditions as for the CP-Trp dyads). Synthesis of PP-Gly. In this case PP was first obtained from photodegradation of CP in deaerated MeOH in the presence of NaOH.21 After eliminating the remaining CP by silica gel chromatography (hexane/ethyl acetate 60/40), the dyad was synthesized from PP as described above for CP-Gly. N-[2(S)-(6-Chloro-9H-carbazol-2-yl)propanoyl]-(S)-tryptophan [(S)-CP-Trp]: 1H NMR (300 MHz, CDCl3) δ (ppm) 1.51 (d, J ) 7.3 Hz, 3H, CH3), 3.17 (m, 2H, CH2), 3.57 (s, 1H, CH3), 3.61 (q, J ) 7.3 Hz, 1H, CH), 4.79 (m, 1H, CH), 5.86 (d, J ) 7.9, 1H, NH), 6.53 (d, J ) 2.3, 1H, CH), 6.90-7.40 (m, 8H, CH aromatic), 7.69 (s, 1H, NH), 7.79 (d, J ) 8.1 Hz, 1H, CH aromatic), 7.91 (br s, 2H, CH aromatic + NH). 13C NMR (75 MHz, CDCl3) δ (ppm) 18.8, 27.6, 47.8, 52.6, 53.1, 110.1, 110.2, 111.5, 112.0, 118.9, 120.0, 120.1, 120.4, 121.1, 122.0, 122.5, 122.9, 124.6, 125.4, 126.4, 127.9, 136.3, 138.4, 139.7, 140.8, 172.6, 174.5. HRMS (EI) calcd for C27H24N3O3Cl 473.1506, found 473.1516. N-[2(R)-(6-Chloro-9H-carbazol-2-yl)propanoyl]-(S)-tryptophan methyl ester [(R)-CP-Trp]: 1H NMR (300 MHz, CDCl3) δ (ppm) 1.54 (d, J ) 7.2 Hz, 3H, CH3), 3.19 (m, 2H, CH2), 3.65 (q, J ) 7.2 Hz, 1H, CH), 3.7 (s, 1H, CH3), 4.97 (m, 1H, CH), 5.93 (d, J ) 7.7, 1H, NH), 6.37 (d, J ) 2.3, 1H, CH), 6.90-7.50 (m, 8H, CH aromatic), 7.75 (s, 1H, NH), 7.90 (d, J ) 8.1, 1H, CH aromatic), 8.05 (br s, 2H, CH aromatic + NH). 13C NMR (75 MHz, CDCl ) δ (ppm) 19.0, 27.7, 47.6, 52.7, 3 52.8, 109.9, 110.0, 111.4, 112.1, 118.9, 120.0, 120.2, 120.4, 121.0, 121.9, 122.4, 123.2, 124.5, 125.4, 126.3, 127.8, 136.2,
Excited State Carprofen/Protein Interactions 138.4, 140.3 140.8, 172.8, 174.3. HRMS (EI) calcd for C27H24N3O3Cl 473.1506, found 473.1501. N-[2(S)-(9H-Carbazol-2-yl)propanoyl]-(S)-tryptophan methyl ester [(S)-PP-Trp]: 1H NMR (300 MHz, CDCl3) δ (ppm) 1.53 (d, J ) 7.3 Hz, 3H, CH3), 3.14 (m, 2H, CH2), 3.57 (s, 3H, CH3), 3.63 (q, J ) 7.3 Hz, 1H, CH), 4.76 (m, 1H, CH), 5.80 (d, J ) 8.1 Hz, 1H, NH), 6.41 (d, J ) 2.3 Hz, 1H, CH), 6.90-7.40 (m, 9H, aromatic CH), 7.47 (br s, 1H, NH), 7.81 (br s, 1H, NH), 7.88 (d, J ) 8.1 Hz, 1H, aromatic CH), 7.99 (d, J ) 7.9 Hz, 1H, CH aromatic). 13C NMR (75 MHz, CDCl3) δ (ppm) 18.7, 27.5, 47.8, 52.8, 52.9, 109.9, 110.0, 111.1, 111.4, 118.9, 119.9, 120.0, 120.1, 120.7, 121.0, 122.5, 122.9, 123.0, 123.3, 126.4, 127.8, 136.3, 138.7, 140.1, 140.2, 172.6, 174.9. HRMS (EI) calcd for C27H25N3O3 439.1896, found 439.1881. N-[2(R)-(9H-Carbazol-2-yl)propanoyl]-(S)-tryptophan methyl ester [(R)-PP-Trp]: 1H NMR (300 MHz, CDCl3) δ (ppm) 1.48 (d, J ) 7.2 Hz, 3H, CH3), 3.09 (m, 2H, CH2), 3.61 (q, J ) 7.2 Hz, 1H, CH), 3.61 (s, 3H, CH3), 4.85 (m, 1H, CH), 5.79 (d, J ) 8.1 Hz, 1H, NH), 6.20 (d, J ) 2.3 Hz, 1H, CH.), 6.80-7.40 (m, 10H, CH aromatic + NH), 7.69 (br s, 1H, NH), 7.88 (d, J ) 8.3 Hz, 1H, CH aromatic), 8.00 (d, J ) 7.7 Hz, 1H, CH aromatic). 13C NMR (75 MHz, CDCl3) δ (ppm) 18.9, 27.6, 47.6, 52.7, 52.7, 109.8, 111.1, 111.3, 119.0, 119.9, 120.0, 120.7, 120.9, 122.4, 122.8, 123.1, 123.3, 126.4, 127.7, 136.2, 139.7, 140.1, 140.2, 172.8, 174.2. HRMS (EI) calcd for C27H25N3O3 439.1896, found 439.1892. N-[2-(6-Chloro-9H-carbazol-2-yl)propanoyl]glycine ethyl ester [CPGly]: 1H NMR (300 MHz, CDCl3) δ (ppm) 1.16 (t, J ) 7.2 Hz, 3H, CH3), 1.56 (d, J ) 7.2 Hz, 3H, CH3), 3.72 (q, J ) 7.2 Hz, 1H, CH), 3.90 (m, 2H, CH2), 4.08 (q, J ) 7.2 Hz, 2H, CH2), 5.86 (br s, 1H, NH), 7.20-7.35 (m, 6H, CH aromatic), 8.05 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3) δ (ppm) 14.7, 19.2, 41.9, 47.7, 61.9, 110.2, 112.0, 120.2, 120.4, 121.3, 122.2, 124.6, 125.5, 126.4, 138.4, 140.0, 140.8, 170.3, 174.8. HRMS (EI) calcd for C19H19N2O3Cl 358.1084, found 358.1073. N-[(2-9H-Carbazol-2-yl)propanoyl]glycine ethyl ester [PPGly]: 1H NMR (300 MHz, CDCl ) δ (ppm) 1.16 (t, J ) 7.2 Hz, 3H, 3 CH3), 1.56 (d, J ) 7.2 Hz, 3H, CH3), 3.72 (q, J ) 7.2 Hz, 1H, CH), 3.89 (m, 2H, CH2), 4.07 (q, J ) 7.2 Hz, 2H, CH2), 5.86 (br s, 1H, NH), 7.10 (dd, J ) 8.1 and 1.13 Hz, 1H, CH aromatic), 7.13-7.35 (m, 5H, CH aromatic), 7.97 (m, 2H, aromatic CH), 8.06 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3) δ (ppm) 14.4, 19.1, 41.9, 47.7, 61.8, 110.0, 111.0, 119.8, 120.0, 120.7, 121.2, 123.1, 123.4, 126.3, 139.1, 140.2, 140.3, 170.2, 175.3. HRMS (EI) calcd for C19H20N2O3 324.1474, found 324.1466. Sample Preparation. The same types of samples were used for laser flash photolysis, steady-state/time-resolved fluorescence, and HPLC experiments; they were prepared as follows: (i) For interactions of CP with HSA, mixtures of racemic, (R)or (S)-CP (25 µM) with HSA (10-25 µM) were dissolved in phosphate buffer and incubated 1 h in the dark; eventually (R)or (S)-IB (4-92 µM) was added in the displacement experiments. (ii) For study of the CP-Trp, PP-Trp, CP-Gly, and PPGly dyads, solutions were prepared in acetonitrile to reach an absorbance of 0.12 at 308 nm. Fluorescence Emission. Mixtures of CP and HSA in PBS were incubated for 30 min in the dark and UVA irradiated. Then, the samples were left 30 min in the dark to allow for any intermediate to decay. Fluorescence emission detected at this step provided information about the formation of the photoproduct. To study drug-protein photobinding, the unbound drug was separated from the protein by using 3 M guanidinium
J. Phys. Chem. B, Vol. 111, No. 2, 2007 425 CHART 1: Structure of Carprofen (CP) and Its Dehalogenated Photoproduct (PP)
hydrochloride solution and Sephadex G-25 columns equilibrated with PBS. The controls included a drug-protein mixture kept in the dark, as well as HSA or carprofen alone with or without irradiation. Photolysis. UVA-irradiated solutions of (R)- or (S)-CP and HSA were studied by HPLC with a reverse phase column C18 Kromasil 100 (5 µm, 25 × 0.4 cm) eluted with methanol/ acetone/water/acetic acid (38/30/30/2, v/v); detection was performed at 330 nm. In the study of racemic CP photolysis, the Chiral Phase Kromasil column (CHI-TBB, 5 µm packing) was used with hexane:tert-butyl methyl ether:acetic acid (60:40:0.1) as the mobile phase. So, CP/HSA mixtures were UVA irradiated and acidified to pH 3 with a diluted HCl solution, and (S)-ketoprofen (KP) was added as the internal standard. CP and KP were extracted with tert-butyl methyl ether, and the organic phase was then studied by HPLC. For the photolysis of the CP-Trp dyads, an equimolar mixture of (R)- and (S)-CP-Trp in acetonitrile was irradiated and studied by HPLC with a reverse phase column C18 Kromasil 100 (5 µm, 25 × 0.4 cm) with acetonitrile/water (1/1, v/v); detection was performed at 300 nm. Results and Discussion Among the numerous commercially available analogues, carprofen (Chart 1) was the nonsteroidal antiinflammatory drug (NSAID) chosen to study stereoselectivity in drug-protein interaction because (i) it preserves its chiral center upon irradiation, as photodecarboxylation is a minor pathway (φ < 0.01), (ii) CP singlet and triplet states are well characterized21 and efficiently populated, and (iii) the dark binding sites and affinity constants of CP stereoisomers have been reported, and the binding process exhibits a significant stereoselectivity.22 Supramolecular Interactions between Triplet Carprofen and HSA. Time-resolved nanosecond laser flash photolysis was performed at 308 nm with an excimer Xe/Cl laser, using argon bubbled PBS solutions of (R)- or (S)-CP (25 µM) with HSA (25 µM). Whatever the stereoisomer considered, only one transient was observed with maximum at λ ) 450 nm (Figure 1A). By comparison with the data previously described for racemic CP in solution, this transient was assigned to the triplettriplet transition.21 It is noteworthy that the carbazolyl radical (λ ) 640 nm) was not detected under these conditions, while it is one of the major transients (together with triplet-triplet absorption and solvated electron) in the transient spectrum of CP in PBS.21 By contrast with the similarity of the transient spectra obtained with the two enantiomers, significant changes were observed in the decays (Figure 1B), where a remarkable stereoselectivity was detected between the lifetimes of the HSA-complexed (R)and (S)-CP. In both cases, the decays monitored at 450 nm appeared to be biphasic with a long-lived major component (A1, τ1) and a short-lived minor one (A2, τ2). As shown in Table 1, both lifetimes of (R)-CP (τ1,R and τ2,R) were longer than those of (S)-CP (τ1,S and τ2,S); the most pronounced stereodifferentiation was observed for the transients with shorter lifetime (τ1,R/
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Figure 1. (A) Transient absorption spectra of (R)- and (S)-CP (25 µM) in the presence of HSA (25 µM) obtained 5 µs after the 308 nm laser pulse. (B) Decays monitored at 450 nm of (R)- and (S)-CP in the presence of HSA.
TABLE 1: Photophysical Properties of the Carprofen Enantiomers in the Presence of Albumin (R)-CP/HSA (S)-CP/HSA
τ1 (µs)a
τ2 (µs)a
A2/A1b
II/Ic
PP (%)d
8.9 2.3
40 24
4.8 10.8
5 10
30 43
a Lifetimes were measured under argon. b A1 and A2 were the components with lifetimes τ1 and τ2. c Distribution of CP in HSA binding sites (site II/site I) calculated from the nK values given in ref 22. d Formation of the photoproduct determined by reverse phase chromatography.
SCHEME 1
τ1,S ≈ 4 vs τ2,R/τ2,S ≈ 2). Another interesting result was that regardless of the component considered, complexation of CP with HSA induced a dramatic lengthening of the lifetimes in comparison with carprofen alone in PBS (τ ca. 0.18 µs). Indeed, as most of the chlorocarbazole derivatives,23 carprofen suffers an efficient self-quenching that would be disfavored by complexation with HSA, where the presence of two (or more) CP molecules in close vicinity is unlikely. This is in agreement with the absence of carbazolyl radical, which is formed upon carprofen self-quenching (Scheme 1) and subsequent deprotonation of CP radical cation. Moreover, another factor contributing to the lengthening of CP decay is probably the more rigid environment provided by the protein. The observation of biphasic decays was remarkable; it was attributed to inclusion of CP in the two binding sites of HSA.22 Actually, dark binding of the CP pure enantiomers to HSA has been described in the literature. The high affinity site (site II) is primarily populated with a slight preference for (S)-CP; by contrast, site I is characterized by a lower affinity. Under the employed experimental conditions, an excellent correlation was observed between the ratio of the lifetime components A2/A1 and the ratio of (R)- and (S)-CP distribution in each HSA binding site determined by other methods (see Table 1, column II/I). Further experiments were performed to confirm the assignment of 3CP components in site I and site II of HSA. First, aerated solutions of albumin (concentration ranging from 10 to 25 µM) and CP (25 µM) were prepared and equilibrated in the dark; then, decays were monitored at 450 nm. As shown in Figure 2, the decrease of HSA concentration induced a noticeable variation of the decay dynamics. Analysis of the signals revealed the progressive appearance of a very short-lived species corresponding to free 3CP (τ ) 0.17 µs) concomitantly with the decrease of the long-lived component. By examining the site II/site I ratio as a function of HSA concentration, it can be concluded that site II is primarily occupied; it is the most
populated when HSA concentration is higher than that of CP. However, the relative population of site II decreases with decreasing concentration of albumin. These results are in excellent agreement with the assignment of the short-lived and long-lived transients to 3CP bound to site I and site II, respectively. By comparing the enantiomers, a stereodifferentiating process was clearly observed, as the dependence of drug distribution on HSA concentration is different for (S)- and (R)CP. The next step was to make use of a site II displacer to further confirm HSA site assignment. For this purpose, (S)-ibuprofen (IB) was chosen for the following reasons: (i) it interacts mainly in site II,24 (ii) its ability to displace CP from site II has been observed by circular dichroism and equilibrium dialysis,24,25 and (iii) it does not absorb light at 308 nm. So, increasing amounts of (S)- or (R)-IB were added to aerated solutions of (R)- and (S)-CP/HSA (25 µM), and transient decays were monitored at 450 nm. Under these conditions, and in parallel with the addition of IB, a decrease of the long-lived component was observed concomitantly with an increase of the free CP fraction and, to a lesser extent, of the short-lived component (Figure 3). All these results are also in complete agreement with the assignment of the long- and short-lived transients to 3CP in site II and site I, respectively. For each CP enantiomer, the differences in the lifetimes must be related to the proper surrounding and stabilization of the drug in the binding sites, as interactions in site I are mainly hydrophobic in nature, while in site II electrostatic forces and hydrogen bonding may also be involved. Another important difference between the two binding sites is the presence of the only tryptophan of the protein in site I (214Trp). Interestingly, it has been previously shown that Trp is the most efficient amino acid able to quench 3CP (kq) 6 × 108 M-1 s-1).26 Thus, the observed shortening of the lifetime τ1 if compared with τ2 could in principle be attributed to the neighborhood of Trp and its participation in an electrontransfer mechanism with 3CP as acceptor. Photoreactivity of the Carprofen/HSA Complexes. In view of the obtained results, the possibility of a stereoselective process during protein photosensitization by CP was considered. In this connection, special attention has been focused on drug-protein photobinding, as formation of covalent photoadducts between drugs and HSA is recognized to be the origin of the well described photoallergy process. Moreover, it has previously been shown that fluorescence coupled with Sephadex filtration is a method of choice to study the photobinding between racemic CP and albumin.26 Steady-state fluorescence measurements on (R)- or (S)-CP solutions in the presence of the protein led to the same emission spectra (Figure 4). Furthermore, similar singlet state lifetimes were obtained by time-resolved fluorescence for the two
Excited State Carprofen/Protein Interactions
J. Phys. Chem. B, Vol. 111, No. 2, 2007 427
Figure 2. (A and C) Transient absorption decays (monitored at 450 nm) of aerated solutions of (R)- and (S)-CP (25 µM) with HSA (10-25 µM). (B and D) Distribution of CP population in site II/site I of HSA as a function of protein concentration.
Figure 3. Variation of the percentage of (R)- and (S)-CP, free or complexed to site I or site II, of albumin as a function of added (R)- and (S)-IB concentration.
stereoisomers in the presence of HSA and for CP alone in PBS (τ ) 1.2 ns). Then, (R)-CP, (S)-CP, or racemic CP (25 µM) were UVA irradiated in the presence of HSA (25 µM) and studied by steady-state fluorescence. The most salient feature was the marked enhancement of the emission spectra of the photomixtures (Figure 4A). (R)-CP gave rise to a structureless spectrum, while (S)-CP showed a more intense and wellstructured spectrum. In the case of the racemic, the shape and
intensity of the bands were intermediate between those of (R)and (S)-CP. Under the same conditions, CP or HSA irradiated alone showed very weak fluorescence (data not shown). Guanidine treatment and sephadex filtration were run to eliminate the noncovalently bound drug or photoproduct. Operating in this way, nonirradiated samples showed no CPrelated fluorescence, in agreement with the complete elimination of the drug by filtration, while CP/HSA irradiated samples gave
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Figure 4. (A) Fluorescence spectra of irradiated and nonirradiated (R)- and (S)-CP (25 µM) in the presence of HSA (25 mM), excitation wavelength: λexc ) 320 nm. (B) Fluorescence emission of sephadex filtered photomixtures of (R)- and (S)-CP/HSA.
Figure 5. Fluorescence spectra of racemic PP (25 µM) in the presence of HSA (25 µM), λexc ) 320 nm.
rise to structureless spectra (Figure 4B) with an intensity somewhat higher for (R)-CP. So, the detected fluorescence of irradiated samples after filtration is indicative of covalent bond formation between the drug and the protein, by contrast with the nonirradiated samples. It is known that irradiation of CP alone in PBS results in a decrease of the fluorescence intensity, due to polymerization of the drug.21 In the presence of HSA, the enhanced intensity of the emission spectrum is indicative of the important role of the protein in the photolysis of CP. Moreover, the different shape and intensity of the emission spectra of the nonfiltered CP/HSA photomixtures as compared with the nonirradiated CP/HSA spectrum can be attributed in part to the presence of the dechlorinated carprofen photoproduct (PP). Indeed, the emission spectrum of PP has been described as ca. 6 times more intense and 10 nm blue-shifted by comparison to the parent drug.21 Concerning photobinding, it appears reasonable that the bound photoproduct corresponds to a dechlorinated derivative of carprofen, as the emission after sephadex filtration is more intense than that of the initial solutions of CP with HSA. To gain more insight into the mechanism of photobinding, interaction of racemic PP with HSA was also studied by fluorescence (Figure 5). Nearly the same spectra were observed before and after UVA irradiation, while sephadex filtration resulted in the disappearance of the emission, evidencing both the stability of PP within HSA and the lack of reaction between PP and the protein. Thus, the chlorine atom appears essential for the process, indicating that C-Cl cleavage is the source of highly reactive radicals that are the key chemical entities responsible for photobinding. To check the possible formation of the dechlorinated photoproduct PP inside HSA and to confirm a stereodifferentiating process, aerated solutions of (S)- and (R)-CP were irradiated in the presence of albumin and studied by HPLC. Only one photoproduct, identified as PP, was detected concomitantly with the consumption of CP. A clear stereodifferentiation was
Figure 6. (R)- and (S)-PP formation upon UVA irradiation of racemic CP in the presence of HSA (left) and the obtained HPLC-chromatographic analogies (right).
CHART 2: Structures of the Dyads CP-Trp, CP-Gly, PP-Trp, and PP-Gly
observed in product formation, as PP was formed ca. 1.5 times more efficiently in the case of (S)-CP/HSA, (Table 1). This experiment was also performed by irradiation of the racemic mixture of CP and HSA followed by chiral HPLC; this secured identical experimental conditions (concentration, number of photons absorbed, etc.) for the two enantiomers. As expected, formation of (S)-PP was 1.4 times more efficient than that of its (R) counterpart (Figure 6), which is in agreement with the relative intensities of the spectra shown in Figure 4 and supports a stereodifferentiation in PP formation. Intramolecular Triplet Excited-State Interactions in Model Carprofen-Tryptophan Dyads. Compounds CP-Trp, CP-Gly, and PP-Gly were prepared by condensation between racemic CP (or its dechlorinated photoproduct PP) and Trp methyl ester or Gly ethyl ester. The dechlorinated bichromophores (R)- and (S)-PP-Trp were obtained by UVA irradiation of (R)- and (S)CP-Trp, respectively. The structures were unambiguously determined by 1H and 13C NMR, as well as by HRMS. Nanosecond laser flash photolysis experiments were performed at 308 nm (excimer Xe/Cl Laser) in deaerated acetonitrile solutions. The nonreactive dyads CP-Gly and PP-Gly were used as reference compounds to determine the intramolecular quenching rate constants. Laser flash photolysis of CP-Gly gave rise to 3CP (λmax) 440 nm, τ ) 3.3 µs) together with small amounts of the longer lived carbazolyl radical absorbing at λ ) 640 nm (Figure 7A). For bichromophoric (S)-CP-Trp and (R)-CP-Trp, in addition to
Excited State Carprofen/Protein Interactions
J. Phys. Chem. B, Vol. 111, No. 2, 2007 429
Figure 7. Transient absorption spectra of deaerated solutions of CP-Gly (A) or (R)-CP-Trp (B).
TABLE 2: Singlet State Properties of the CP-Derived Dyads in Nitrogen-Bubbled Acetonitrile CP CP-Gly (R)-CP-Trp (S)-CP-Trp CZ PP-Gly (R)-PP-Trp (S)-PP-Trp a
Figure 8. Transient decays of (R)- and (S)-CP-Trp monitored at 450 nm. 3CP,
a long-lived species with λmax ca. 580 nm was also detected. Figure 7B shows the case for (R)-CP-Trp; similar spectra (not shown) were obtained for (S)-CP-Trp. Under aerobic conditions, 3CP was quenched by oxygen, while the 580 nm absorbing species remained almost unaffected, suggesting that it corresponds to the Trp radical cation (Trp+°), with characteristic spectrum peaking at λ ) 580 nm.27 The triplet lifetimes of the (R)- and (S)-bichromophores were 2.4 and 3.0 µs, respectively (see decays in Figure 8). This shortening of the lifetime (if compared with that of 3CP-Gly), associated with detection of the Trp radical cation, strongly supports an electron transfer from Trp to the CP triplet state. By applying the relationship kq) 1/τ - 1/τo, quenching rate constants of 1.1 × 105 and 3.0 × 104 s-1 were calculated for (R)- and (S)-CP-Trp; this revealed a significant stereodifferentiation in the intramolecular quenching process derived from the ratio k(R)-CP-Trp/k(S)-CP-Trp) 3.7. Bichromophoric dyads containing the dehalogenated photoproduct of CP, namely (R)- and (S)-PP-Trp, as well as PP-Gly, were also studied to detect a possible intramolecular photoreaction. As shown in Figure 9, only 3PP and traces of carbazolyl radical were detected as transient species. In accordance with the absence of Trp radical cation, very similar triplet lifetimes
λf (nm)
ES (kJ‚mol-1)
τf (ns)
φf
345 345 345 345 330 335 335 335
346 346 346 346 362a 357 357 357
1.45 1.58 1.55 1.53 15.6 14.5 14.4 14.5
0.068 0.066 0.068 0.069 0.42a 0.34 0.39 0.37
Similar data were described for PP.21
of ca. 10 µs were observed for the three compounds, ruling out an electron-transfer photoprocess between Trp and PP. Intermolecular quenching of (R)- and (S)-CP by Trp methyl ester was also performed in acetonitrile, and a similar rate constant of 5.4 × 108 M-1 s-1 was determined for the two stereoisomers. So, it seems that the conformational constraints and the limited degrees of freedom characteristic of the dyads are essential for the occurrence of a stereodifferentiating process. Photoreactivity of the Dyads. In a first stage, fluorescence measurements were used to establish a correlation between the photoreactivity of CP in the covalently linked dyads and that of the CP/HSA complexes. As the emission properties of (R)and (S)-CP-Trp were unknown, these compounds were submitted to a systematic steady-state and time-resolved fluorescence study; similar measurements were performed on (R)-PP-Trp, (S)-PP-Trp, CP-Gly, and PP-Gly, for comparison. The obtained data are summarized in Table 2. As expected, all the CP-derived dyads exhibited a parallel behavior, very similar to that of the parent CP. This is in agreement with the lack of singlet excitedstate interaction in the CP/HSA systems (see above). Likewise, the quantum yields and lifetimes of the PP-based dyads did not differ significantly from those of carbazole, which was taken as reference. Clearly, the presence of a chloro substituent resulted in a much less efficient and shorter lived fluorescence.
Figure 9. Transient absorption spectra of PP-Gly (A) and (R)-PP-Trp (B) in deaerated acetonitrile solutions.
430 J. Phys. Chem. B, Vol. 111, No. 2, 2007
Lhiaubet-Vallet et al. stereodifferentiation found in the interaction between excited CP and HSA; this is reflected in the markedly different triplet lifetimes of the two CP enantiomers in both binding sites, especially in the Trp-containing site I. Accordingly, a significant stereodifferentiation is also observed in the intramolecular quenching of 3CP by Trp in the model dyads. In general, the prevailing photochemical process is cleavage of the C-Cl bond; this leads to stereoselective formation of the dehalogenated photoproduct PP both in the CP/HSA complex and in the dyads. Besides, some stereodifferentiation is also detected in the covalent photobinding of CP to the protein. These results are relevant in the context of CP-mediated photoallergy.
Figure 10. Fluorescence spectra of nitrogen-bubbled solutions of (R)and (S)-CP-Trp before and after UVA irradiation.
Acknowledgment. Financial support by the Spanish government (CTQ2004-03811) and the CSIC (postdoctoral I3P contract to V.L.-V.) is gratefully acknowledged. References and Notes
Figure 11. Time-dependent formation of (R)- and (S)-PP-Trp by irradiation of an equimolar mixture of (R)- and (S)-CP-Trp in deaerated acetonitrile.
After establishing the emission behavior of the dyads, a steady-state photolysis study was undertaken. Thus, when acetonitrile solutions of (R)- and (S)-CP-Trp were UVA irradiated, the emission spectra were markedly enhanced and became better resolved (Figure 10), in excellent agreement with the results obtained within the protein microenvironment (Figure 4A). This effect was somewhat more marked in the case of (R)CP-Trp, revealing (as in the CP/HSA complexes) a significant stereodifferentiation in the photoreactivity of the dyads. In view of the higher emission quantum yields of all the PP derivatives (Table 2), the observed increase of the fluorescence intensity upon irradiation strongly suggested a photodehalogenation process. To check this possibility, the reaction was followed by HPLC. Moreover, to ensure that (R)- and (S)-CPTrp were receiving the same number of photons, an equimolar mixture of the two isomers was employed. As a matter of fact, the only photoproducts detected were the corresponding (R)or (S)-PP-Trp isomers. Again, a clear stereodifferentiation was observed in photoproduct formation, as shown in Figure 11, with (R)-CP-Trp reacting somewhat faster. This is the same trend exhibited by the fluorescence enhancement and the triplet quenching rate process. Conclusion The appearance of two components with different lifetimes in the triplet decay of CP within HSA is explained by complexation of the drug to the two protein binding sites. The shorter lifetime of site I-bound CP is mainly attributed to electron-transfer quenching by the only Trp unit present in HSA. This assignment is confirmed by using IB in the site IIdisplacement experiment and also by the intramolecular quenching observed in model CP-Trp dyads, whose laser flash photolysis reveals formation of the expected radical cation (Trp+°) absorbing at ca. 580 nm. A remarkable result is the
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