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Reaction Dynamics of Excited 2-(3-Benzoylphenyl)propionic Acid (Ketoprofen) with Histidine Tadashi Suzuki,* Takeshi Okita, Yohei Osanai, and Teijiro Ichimura Department of Chemistry and Materials Science, Tokyo Institute of Technology, Ohokayama, Meguro, Tokyo 152-8551, Japan ReceiVed: July 18, 2008; ReVised Manuscript ReceiVed: September 8, 2008
Photoreaction dynamics of 2-(3-benzoylphenyl)propionic acid (ketoprofen, KP), one of nonsteroidal antiinflammatory drugs, with histidine in a phosphate buffer solution (pH 7.4) was investigated with the laser flash photolysis. The deprotonated form of KP (KP-) was decarboxylated via UV laser excitation to form a carbanion. It was found that histidine accelerates the protonation reaction of the carbanion to 3-ethylbenzophenone ketyl biradical (3-EBPH) for the first time. The experimental results of the photoreaction of KP with alanine as well as the photoreaction of KP with 4-methylimidazole (a part of the side chain of histidine) in methanol, clearly showed that the protonated form of histidine is a key species for the protonation reaction of the carbanion. These series of the initial reactions should result in the occurrence of photosensitization in vivo. The reaction mechanism was discussed in detail. SCHEME 1
1. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used in the clinical treatment. In the past decade, the photochemistry of the NSAIDs has been the subject of considerable interest since their photosensitization was reported.1,2 2-(3Benzoylphenyl) propionic acid (ketoprofen, abbreviated as KP, Scheme 1), one of the most widely used NSAIDs, is a photoxic and photoallergic agent1-8 and thus clinically evokes photocontact dermatitis. The photoreaction of KP in solution has been extensively studied with product analysis,9,10 picosecond/nanosecond transient absorption measurements,11-13 time-resolved laser-induced optoacoustic technique,14 and quantum chemical calculations.15,16 Excited KP- in phosphate buffer yields a carbanion, structurally resonant structure with 3-ethylbenzophenone ketyl biradical anion (3-EBP-), through a photodecarboxylation reaction.14 The reported quantum yield of the reaction was as large as ΦPDC ) 0.75.9 At pH < 7.4, a proton was quickly transferred from the solvent to 3-EBP-, yielding 3-ethylbenzophenone ketyl biradical (3-EBPH). Subsequent intramolecular hydrogen transfer of 3-EBPH produced 3-ethylbenzophenone (3-EBP) as a final product. At pH > 7.4, the carbanion was directly protonated from H2O to produce 3-EBP. This final product of 3-EBP was characterized by liquid chromatography mass spectrometry.10 In our previous report, the photochemistry of KP in the presence of triethylamine (TEA) in methanol has been investigated as a prototype of KP reaction with amines in vivo with transient absorption and two-color excitation emission techniques.17,18 In the presence of a hydrogen donor such as methanol, KP underwent hydrogen abstraction to yield ketyl radical (KPH), exhibiting photochemical behavior similar to benzophenone. Since KP is an acid, acid-base equilibrium, KP + TEA h KP+ TEAH+, is established. The reaction dynamics of KP- in methanol in the presence of TEA turned out to be similar to that in phosphate buffer solution. The excited KP- was rapidly decarboxylated to yield carbanion. It was concluded that the * To whom correspondence should be addressed. Phone/Fax: +81-35734-2331. E-mail:
[email protected].
carbanion reacted with TEAH+ (proton donor) to produce 3-EBPH. Furthermore, it became clear that 3-EBPH and TEA formed a hydrogen bonding complex of 3-EBPH...TEA. The reaction mechanism is summarized in Scheme 2. These experimental results suggest that amines play an important role in initial photoreaction of KP, and some basic biomolecule may be a key species for the radical formation and reaction in vivo, probably followed by appearance of toxicity. KP is a chiral molecule with an asymmetric carbon. To gain the detailed information on KP photosensitization in biosystems, KP-protein complex formation and stereoselective reaction were studied.19-24 Monti and co-workers reported the binding constant of KP to two major binding sites of the bovine serum albumin by circular dichromism and fluorescence measurements.22,23 They showed the site dependent photoreactivity of KP in the protein sites; however, the reaction process could not be clarified by the time-resolved spectroscopic approach. These results suggest that the reaction mechanism in a protein is rather complicated. A protein consists of twenty kinds of amino acids. To understand the initial photoreaction of KP with a protein in vivo, which is a trigger for the production of an allergen, it is important to obtain information on its reactivity with amino acids. Histidine, arginine, and lysine are known to be basic amino acids. The structure of histidine, having an imidazolium
10.1021/jp8063382 CCC: $40.75 2008 American Chemical Society Published on Web 10/31/2008
Photoreaction of Ketoprofen with Histidine
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SCHEME 2: Photoreaction of KP with TEA in Methanol Solution or in Phosphate Buffer Solution
side chain, is quite sensitive to the microenvironmental physiologic pH because of its pKa (6.04). It affects enzyme function of a protein. In our earlier work,25,26 the reaction of excited KP with histidine in the phosphate buffer solution was reported. We showed that the KP is quenched by histidine. In this study, reaction dynamics of excited KP with histidine in the phosphate buffer solution (pH 7.4) was investigated in detail via laser flash photolysis technique to elucidate an initial reaction scheme of KP with protein in vivo after photoexcitation. 2. Experimental Section An experimental setup of the transient absorption spectrum measurement has been described elsewhere.17 Briefly, a XeCl excimer laser (Lamda Physik, COMPex 102; 308 nm, 200 mJ, 20 ns pulse duration) and a cw Xe flash lamp (Ushio, UXL300DO; 300 W) were used as an exciting light source and a monitoring light source, respectively. The laser beam was introduced into a sample flow cell (NSG, T-59FL-10; 10 mm optical path length). The monitoring light passed through a monochromator (Nikon, P250) and was detected with a photomultiplier tube (Hamamatsu Photonics, R928). The signal was captured by a digital oscilloscope (Sony Tektronix, TDS380P; 2 GS/s, 400 MHz), and transferred to a personal computer. The signal was averaged over 30 laser shots to improve the S/N ratio. Fresh sample solution flowed in the flow cell during the measurements to avoid the contamination due to photoproducts. All measurements were carried out at room temperature. Absorption spectra were measured with a double beam spectrophotometer (JASCO Ubest V-550). KP (Sigma-Aldrich; purity 99.9%), L-histidine (His; Wako Chemical, GR grade), L-alanine (Ala; Kanto Chemical, GR grade), and 4-methylimidazole (MI; Kanto Chemical, GR grade) were used without further purification. Distilled water (HPLC grade) and methanol (GR grade), purchased from Kanto Chemical, were used as solvents from freshly opened bottles.
Phosphate buffer solution (Kanto Chemical, 1/15 M) was pH 7.4. All sample solutions were carefully prepared to maintain a pH of 7.4 and were deaerated by argon gas (purity 99.95%) saturated by a solvent vapor for half an hour before use. 3. Results and Discussion 3.1. Photoreaction of KP with Histidine in the Phosphate Buffer Solution. In the phosphate buffer solution of pH 7.4, almost all KP exists in the deprotonated form of KP, KP-, in the ground-state because of its acidity (pKa ) 4.7).11-14 Figure 1 shows the transient absorption spectra of KP (0.70 mM) in the phosphate buffer solution (pH 7.4) obtained with the XeCl excimer laser irradiation. An intense band at near 600 nm appeared immediately after the laser pulse. This band results from a carbanion. Figure 2a shows the decay time profile of transient absorption monitored at 600 nm. The lifetime was determined to be 93 ns with the single-exponential fitting
Figure 1. Transient absorption spectra of KP in the phosphate buffer solution at pH 7.4 obtained immediately after the laser (b), at 40 ns (9), 100 ns (2), and 1.7 µs (().
15214 J. Phys. Chem. B, Vol. 112, No. 47, 2008
Suzuki et al.
k0 + kr[His] kr [3-EBPH] ) ) 1 + [His] [3-EBPH]0 k0 k0
Figure 2. Time profiles of transient absorption monitored at 600 nm (a) without and (b) with histidine (20 mM) in the phosphate buffer solution (pH 7.4). The inset denotes Stern-Volmer plots of the deactivation rate constant of the carbanion against the concentration of histidine. The carbanion disappeared rapidly with histidine addition. The apparent quenching rate constant was determined to be 2.9 × 108 M-1s-1.
analysis of the decay profile. At 1.7 µs after the laser, new absorption band was observed at near 525 nm. Although this spectral feature is similar to that of the benzophenone ketyl type radical, as shown in Introduction it was assigned to 3-EBPH, which produced by protonation reaction of a carbanion from a solvent.1,14 3-EBPH has a lifetime of several µs to produce 3-ethylbenzophenone as a final product through the intramolecular hydrogen atom transfer reaction. The transient absorption spectrum of KP with several concentrations of His in phosphate buffer solution (pH 7.4) was measured. Figure 2b shows the decay time profile of transient absorption monitored at 600 nm with 20 mM of His. An acceleration of the absorption intensity decay is clearly observed upon addition of His. The single-exponential decay fitting analysis was carried out at each concentration of His to obtain the quenching rate constant of the carbanion by His. Plots of the His concentration dependence of the decay rate constant are shown in the inset of Figure 2. A good linear relation was found. The slope of the straight line, namely, the apparent bimolecular quenching rate constant of the carbanion by His, 8 -1 -1 by the least-squares’ kapp q was obtained to be 2.9 × 10 M s fitting method. Here, we will consider the reaction process of the carbanion with histidine. In the previous report,17 we clarified the reaction mechanism of the carbanion with TEA in methanol. The carbanion was protonated by HN+(C2H5)3 (proton donor), which was produced through the acid-base equilibrium of KP and TEA in the ground state, to form 3-EBPH (see Scheme 2). Figure 3 shows time profiles of transient absorption monitored at 525 nm with and without 20 mM of His. The fast and slow decaying components were due to the carbanion and 3-EBPH, respectively. The profiles clearly indicate that the amount of 3-EBPH increases with His. To estimate the amount of 3-EBPH, the absorption intensities at 525 nm of carbanion (Acarbanion) and 3-EBPH (A3-EBPH) at each concentration of His (see Figure 3) were compared. The ratio of the A3-EBPH/Acarbanion values with and without His, (A3-EBPH/ Acarbanion)/(A3-EBPH/Acarbanion)0, equals to [3-EBPH]/[3-EBPH]0, where [3-EBPH]0 and [3-EBPH] are the initial concentration of 3-EBPH without and with His, respectively. Figure 4 shows the His concentration dependence of the [3-EBPH]/[3-EBPH]0 value. The [3-EBPH]/[3-EBPH]0 value was found to increase linearly with [His]. The [3-EBPH]/[3-EBPH]0 value was rewritten as follows.
(1)
Here, k0 and kr denote the formation rate constants of 3-EBPH from the carbanion without and with His, respectively. The slope of the best-fitted straight line in Figure 4, namely, the ratio of the apparent rate constants krapp/k0app, was successfully determined to be 7.16. The apparent reaction rate constant, krapp, was estimated to be 7.7 × 107 M-1s-1, assuming that the k0app value equals to 1.07 × 107 M-1s-1, the deactivation rate constant of the carbanion without His. Therefore, the ratio of krapp/kqapp denotes the probability of the 3-EBPH formation from the carbanion with His by collision. So, the reaction quantum yield of the carbanion with His was roughly estimated to be 0.27. It is clarified that proton transfer reaction takes place between the carbanion and His. His in the pH 7.4 solution has two kinds of proton-donating sites (Scheme 3): the protonated N-terminal site (-NH3+, site I) and the side chain of imidazole (site II). In addition, a minor form of protonated His should exist in the phosphate buffer solution (pH 7.4) because pKa ) 6.04 of the side chain (site III).27 To further clarify the reaction mechanism of KP with His in detail, the transient absorption spectrum of KP with 20 mM of alanine in the phosphate buffer solution (pH 7.4) was measured. Alanine in the phosphate buffer solution has a protonated form of the amino group (the protonated N-terminal site, pKa 9.87);27 however, no photoreaction of KP with alanine was observed. So, one can conclude that the side chain of His is necessary for the proton transfer reaction of KP with His. To confirm this idea, photoreaction of excited KP and
Figure 3. Time profiles of transient absorption monitored at 525 nm (a) without and (b) with histidine (20 mM) in the phosphate buffer solution (pH 7.4). Two decay components were observed. The P is Acarbanion and the Q is A3-EBPH pre-exponential factors denote the amount of the carbanion and 3-EBPH, respectively. See text for details.
Figure 4. Plots of [3-EBPH]/[3-EBPH]0 against the concentration of histidine. The relative formation yield of 3-EBPH increases with histidine.
Photoreaction of Ketoprofen with Histidine
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SCHEME 3: Structures of His in the Phosphate Buffer Solution of pH 7.4
4-methylimidazole (MI), which is the side chain of His, was investigated. 3.2. Reaction Dynamics of Excited KP with 4-Methylimidazole in Methanol. The reaction mechanism of KP with MI, which has the same structure as the side chain of His, was investigated. First, the absorption spectra of KP (1.25 mM) with several concentrations of MI (0-50 mM) in methanol were measured. The absorption spectrum gradually varied with the concentration of MI. In the same manner as the previous report,17 the equilibrium constant of the acid-base equilibrium KP + MI h KP- + MIH+
K)
[KP-][MIH+] [KP][MI]
Figure 6. Plots of the deactivation rate constant of the carbanion against the concentration of MI, [MI]. Linear Stern-Volmer quenching was not observed.
(2)
was estimated. The absorbance, A(λ), at a given wavelength λ is written as λ λ + λ A(λ) ) εKP [KP]d + ελMI[MI]d + εKP -[KP ]d + εMIH+[MIH ]d . λ λ λ λ λ [KP]0d + ελMI[MI]0d + (εKP ) εKP - - εKP + εMIH+ - εMI)d × .
([KP]0 + [MI]0)K - √([KP]0 - [MI]0)2K2 + 4K[KP]0[MI]0 (3) 2(K - 1) λ , ελ , ελ -, and ελ + represent the absorption where εKP MI KP MIH coefficients of KP, MI, KP-, and MIH+, respectively. [KP]0 and [MI]0 are initial concentration of KP and MI, respectively. Under this experimental condition, the initial concentration of 308nm 308nm value are 326,17 KP was 1.25 mM. The ε308nm KP , εKP- , and εMI 17 -1 -1 714, and 3.6 M cm , respectively. From the least-squares’ method with eq 3, the K value was successfully determined to be 0.236. Figure 5 shows the concentration of KP, KP-, and MIH+. From this estimation, the concentrations of KP and KP- changed considerably with the amount of added MI. Such a drastic
Figure 5. Concentration of KP, KP-, and MIH+ against added MI. The initial concentration of KP was 1.25 mM and the acid-base equilibrium constant K was obtained to be 0.236.
Figure 7. Plots of the deactivation rate constant of the carbanion against the concentration of MIH+, [MIH+]. The linear relation between the rate constant and [MIH+] was clearly observed. The quenching rate constant was determined to be 1.42 × 1010 M-1s-1. The reaction occurs effectively at the diffusion-controlled rate.
TABLE 1: The Net Quenching Rate Constant of the Carbanion, kqnet, and the Reaction Rate Constant, krnet quencher
solvent
HisH+
phosphate buffer (pH 7.4) methanol methanol
MIH+ TEAH+
kqnet /M-1s-1 krnet /M-1s-1 6.7 × 109 1.42 × 1010 1.1 × 1010
1.8 × 109
this work this work ref 17
concentration change of each component in the ground-state will affect the reaction dynamics in the excited-state of KP or KP-. The transient absorption spectra of KP (1.25 mM) with several concentrations of MI (1-50 mM) in methanol were measured with 308 nm excitation. The transient absorption spectrum was similar to that in the presence of His (see Figure 1). The absorption peak that appears at near 600 nm immediately after the laser excitation was due to the carbanion, which was produced by decarboxylation of 3KP-*. Figure 6 shows plots of the decay rate constant against the concentration of added MI. Linear Stern-Volmer quenching was not observed. Instead, the rate constant increased with [MI] and approached an asymptote at high [MI]. With a known value for the equilibrium constant K the amount of MIH+ was estimated. Surprisingly, good linear relation was obtained (Figure 7). The reaction rate constant was successfully determined to be kqnet ) 1.42 × 1010 M-1s-1. This value was nearly equal to the diffusion-controlled rate of methanol of 1.2 × 1010 M-1s-1 at 298 K.28 In conclusion, the protonated form of MI (MIH+) is a key species for photoreaction of KP-. 3.3. Estimation of Net Quenching Rate Constant of Carbanion by His and Reaction Dynamics of KP in a Protein. In the previous section, it becomes clear that not MI but the protonated form of MI (MIH+) accelerates the reaction
15216 J. Phys. Chem. B, Vol. 112, No. 47, 2008 of the carbanion to 3-EBPH. Therefore, in the case of photoreaction of KP with His, HisH+ should be involved in the reaction. In Section 3.1, the apparent bimolecular quenching rate constant of the carbanion by His was described. To obtain the net quenching rate constant, the concentration of HisH+ was estimated ([His]/[HisH+] ) 23:1). Finally, we successfully obtained the net quenching rate constant, kqnet, to be 6.7 × 109 M-1s-1. This value nearly equals to the diffusion controlled rate constant of water of 7.4 × 109 M-1s-1 at 298 K.28 Moreover, the krnet value was 1.8 × 109 M-1s-1. These results are summarized in Table 1. In conclusion, it becomes clear that the minor species of His in the pH 7.4 phosphate buffer solution, namely, HisH+, is the most important species for the photoreaction of KP and the reaction rate of the carbanion and HisH+ is as large as the diffusion controlled rate. Other basic amino acids, arginine and lysine, also quench effectively the carbanion and form 3-EBPH.29 The side chain of the amino acids was found to be quite important for understanding the photochemistry of KP. In the hydrophobic environment, KP will produce a KPH radical via a hydrogen abstraction reaction by the UV photolysis, while in the hydrophilic condition KP- will form 3-EBPH via a decarboxylation reaction, which will accelerate by basic amino acids in a protein. The photoreaction of KP in a protein has been investigated by several groups as described in the Introduction.19-24 However, it is difficult to clarify the reaction process because the transient spectra are so complicated due to spectral overlap of transients. The spectra of KPH and 3-EBPH are similar each other,17 and the spectrum of a counter radical formed by a hydrogen abstraction by 3KP* such as a tryptophan radical is also superimposed.23 The spectrum of a carbanion has not been observed in a protein. But it may result from the ultrafast quenching reaction with the protonated histidine neighboring the carbanion. The reaction dynamics described in this article will contribute to the elucidation of the photoreaction mechanism of KP in the protein, and will give us new information of NSAIDs on photosensitization in vivo. References and Notes (1) Bosca, F.; Miranda, M. A. J. Photochem. Photobiol., B 1998, 43, 1. (2) Bosca, F.; Marin, M. L.; Miranda, M. A. Photochem. Photobiol. 2001, 74, 637. (3) Artuso, T.; Bernadou, J.; Meunier, B.; Piette, J.; Paillous, N. Photochem. Photobiol. 1991, 54, 205.
Suzuki et al. (4) Radschuweit, A.; Ruttinger, H.-H.; Nuhn, P.; Wohlrab, W.; Huschka, Chr. Photochem. Photobiol. 2001, 73, 119. (5) Lhiaubet, V.; Paillous, N.; Chouini-Lalanne, N. Photochem. Photobiol. 2001, 74, 670. (6) Nakajima, A.; Tahara, M.; Yoshimura, Y.; Nakazawa, H. Chem. Pharm. Bull. 2007, 55, 1431. (7) Imai, S.; Atarashi, K.; Ikesue, K.; Akiyama, K.; Tokura, Y. J. Dermatol. Science 2006, 41, 127. (8) Atarashi, K.; Kabashima, K.; Akiyama, K.; Tokura, Y. J. Dermatol. Science. 2007, 47, 151. (9) Costanzo, L. L.; Guidi, G. D.; Condorelli, G.; Cambria, A.; Fama, M. Photochem. Photobiol. 1989, 50, 359. (10) Bosca, F.; Miranda, M. A.; Carganico, G.; Mauleon, D. Photochem. Photobiol. 1994, 60, 96. (11) Martinez, L. J.; Scaiano, J. C. J. Am. Chem. Soc. 1997, 119, 11066. (12) Cosa, G.; Martinez, L. J.; Scaiano, J. C. Phys. Chem. Chem. Phys. 1999, 1, 3533. (13) Monti, S.; Sortino, S.; Guidi, G. D.; Marconi, G. J. Chem. Soc., Faraday Trans. 1997, 93, 2269. (14) Borsarelli, C. D.; Braslavsky, S. E.; Sortino, S.; Marconi, G.; Monti, S. Photochem. Photobiol. 2000, 72, 163. (15) Musa, K. A. K.; Matxain, J. M.; Eriksson, L. A. J. Med. Chem. 2007, 50, 1735. (16) Lhiaubet, V.; Gutierrez, F.; Penaud-Berruyer, F.; Amouyal, E.; Daudey, J.-P.; Poteau, R.; Chouini-Lalanne, N.; Paillous, N. New J. Chem. 2000, 24, 403. (17) Suzuki, H.; Suzuki, T.; Ichimura, T.; Ikesue, K.; Sakai, M. J. Phys. Chem. B 2007, 111, 3062. (18) Suzuki, T.; Suzuki, H.; Ichimura, T. Photomed. Photobiol. 2005, 27, 13. (19) Bosca, F.; Carganico, G.; Castell, J. V.; Gomez-Lechon, M. J.; Hernandez, D.; Mauleon, D.; Martinez, L. A.; Miranda, M. A. J. Photochem. Photobiol., B 1995, 31, 133. (20) Lhiaubet-Vallet, V.; Encinas, S.; Miranda, M. A. J. Am. Chem. Soc. 2005, 127, 12774. (21) Abad, S.; Bosca, F.; Domingo, L. R.; Gil, S.; Pischel, U.; Miranda, M. A. J. Am. Chem. Soc. 2007, 129, 7407. (22) Monti, S.; Manoli, F.; Sortino, S.; Morrone, R.; Nicolosi, G. Phys. Chem. Chem. Phys. 2005, 7, 4002. (23) Monti, S.; Manet, I.; Manoli, F.; Morrone, R.; Nicolosi, G.; Sortino, S. Photochem. Photobiol. 2006, 82, 13. (24) He, Y.-Y.; Ramirez, D. C.; Detweiler, C. D.; Mason, R. P.; Chignell, C. F. Photochem. Photobiol. 2003, 77, 585. (25) Suzuki, T.; Okita, T.; Ichimura, T. Photomed. Photobiol. 2006, 28, 17. (26) Suzuki, T.; Okita, T.; Ichimura, T. Photomed. Photobiol. 2007, 29, 25. (27) Lide, D. R. Handbook of Chemistry and Physics, 84th ed.; CRC Press: Boca Raton, FL, 2003. (28) Murov, S. L.; Carmicheal, I.; Hug, G. L. Handbook of Photochemistry, ReVised and Expanded, 2nd ed.; Marcel Dekker: New York, 1993. (29) Suzuki, T.; Osanai, Y.; Ichimura, T. Tokyo Institute of Technology. Unpublished work.
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