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Electrochemical behavior of a Cypridina luciferin analogue in acetonitrile solutions in the presence of proton acceptors
www.elsevier.nl/locate/jelechem Journal of Electroanalytical Chemistry 476 (1999) 46 – 53
Electrochemical behavior of a Cypridina luciferin analogue in acetonitrile solutions in the presence of proton acceptors Takeyoshi Okajima, Koichi Tokuda, Takeo Ohsaka * Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226 -8502, Japan Received 2 March 1999; received in revised form 22 June 1999; accepted 10 August 1999
Abstract The electrochemical redox behavior of the Cypridina luciferin analogue, 6-(4-methoxyphenyl)-2-methylimidazo[1, 2-a]pyrazin3(7H)-one (MCLA) has been examined in acetonitrile solutions containing 2,6-lutidine or water using cyclic and hydrodynamic voltammetry and double potential-step chronoamperometry. The conjugate base of MCLA (MCLA−) is oxidized in two one-electron steps to the radical species (MCLA) and further to the carbocation species (MCLA+). The radical – radical coupling of MCLA yields the corresponding dimer ((MCLA)2), which is in equilibrium with MCLA. MCLA+ undergoes the addition of a water molecule to yield a hydroxylated form (MCLAOH) of luciferinol type. The whole mechanism of the electrochemical redox reactions of MCLA− coupled with these chemical reactions has been found to be essentially the same as that proposed for the chemical redox reactions of Cypridina luciferin and its analogues, and in addition the kinetic data concerning the dimerization of MCLA, the dissociation of (MCLA)2 and the hydroxylation of MCLA+ have been estimated together with the diffusion coefficient of MCLA−. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Cypridina luciferin analogues; Redox reactions; Dimerization; Hydroxylation; Cyclic voltammetry; Double potential-step chronoamperometry; Hydrodynamic voltammetry
1. Introduction The present paper presents the electrochemical behavior of 6-(4-methoxyphenyl)-2-methylimidazo[1,2a]pyrazin-3(7H)-one (MCLA), which is one of the analogues of Cypridina hilgendorfii luciferin [1], in acetonitrile media.
Cypridina luciferin reacts with molecular oxygen (3O2), and consequently emits light in the presence of Cypridina luciferase [2–4]. This system is of great interest, because it is the simplest system of the enzymatic * Corresponding author. Tel.: +81-45-924-5404; fax: + 81-45-9245489. E-mail address: [email protected] (T. Ohsaka)
reactions found in bioluminescence. Goto et al. isolated Cypridina luciferin in a crystalline state and elucidated its structure completely [2]. They also prepared some analogues of luciferin and examined their chemiluminescence properties. In order to clarify the reaction mechanisms of autoxidation of luciferin in the absence of the luciferase, Goto and co-workers investigated the chemical redox reactions of Cypridina luciferin and its analogues by use of oxidizing agents ([Fe(CN)6]3 − , PbO2 and DPPH (2,2-diphenyl-1-picryl hydrazyl)) and reducing agents (Na2S2O4 and NaBH4) in dimethyl sulfoxide, water, various alcohols and water+ alcohol mixtures in the absence of Cypridina luciferase [5,6]. According to the mechanism proposed by them (Scheme 1), the one-electron oxidation of luciferin (L) gives a radical species (L), which is in equilibrium with its dimer species, biluciferyl (LL). L is reduced reversibly with Na2S2O4 to L. L is further oxidized with DPPH to luciferinol and its derivatives (LOR), which can be reduced with Na2S2O4 (or NaBH4) to L. LOR is also generated by two-electron oxidation of L with
0022-0728/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 9 9 ) 0 0 3 6 2 - 9
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
PbO2. No quantitative kinetic investigation on the redox and chemical reactions of luciferin has been reported. Recently, the mechanism and kinetics of autoxidation of its analogue, 2-methyl-6-phenylimidazo[1,2-a]pyrazin3(7H)-one (CLA) have been examined, based on the chemiluminescence measurements, in aqueous solutions of various pHs under aerobic conditions [7,8]. The aim of the present study is to examine the mechanisms of the electrochemical redox reactions of MCLA and the ensuing chemical reactions (i.e. the dimerization of the one-electron oxidation product (MCLA), the dissociation of the dimer ((MCLA)2), and the hydroxylation of the two-electron oxidation product (MCLA+) of MCLA) in acetonitrile media and to obtain the related quantitative kinetic information. The experiments were limited to those in the presence of proton acceptors under a N2 atmosphere to avoid the complexity of the situation resulting from the electrochemical redox reactions of MCLA itself, and the chemical reactions of MCLA and its electrolysis products with molecular oxygen or its electrolytic reduction products (e.g. superoxide ion: O− 2 and H2O2) [9 – 13]. An investigation of the electrochemical behavior of the deprotonated MCLA is of interest in relation to the bioluminescence of Cypridina luciferin. In the proposed mechanisms of bioluminescence, a conjugate base of Cypridina luciferin is essential to the initial reaction. The results obtained confirmed the mechanism proposed by Goto et al. for the chemical redox reactions of luciferin, and in addition the kinetic data concerning the dimerization of MCLA, the dissociation of (MCLA)2, and the hydroxylation of MCLA+ were estimated together with the diffusion coefficient of the deprotonated species (MCLA−) as well as the number of electrons involved in its electrode reaction.
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2. Experimental
2.1. Reagents MCLA (Tokyo Kasei Chemicals Industries) was a specially prepared analytical reagent and was used without further purification. Acetonitrile (AN) of reagent grade was obtained from Kanto Chemical and was used after drying with molecular sieves 4A1/16 (Wako Pure Chemicals Industries). Tetraethylammonium perchlorate (TEAP, Tokyo Kasei) as a supporting electrolyte, and 2,6-dimethylpyridine (2,6-lutidine) (Kanto) as a proton acceptor were used as received. Water was purified by passage through a Millipore Milli-Q purification train. Basal-plane pyrolytic graphite (BPG, Union Carbide) and glassy carbon (GC, Tokai Carbon) were used as the working electrodes. BPG (area: 1.8× 10 − 1 cm2) and GC (area: 7.9 × 10 − 3 cm2) disk electrodes were prepared and pre-treated before use as described previously [14].
2.2. Apparatus and procedures The electrochemical cell and its components were the same as reported previously [15]. BPG and GC disk electrodes were used as the working electrodes, a spiral platinum wire as the auxiliary electrode, and a sodium chloride saturated silver silver chloride electrode (Ag AgCl NaCl(sat)) and Ag wire as the reference electrodes. Cyclic voltammetry in the range of potential sweep rates from 2 to 500 mV s − 1 was carried out using a PS-07 polarization unit (Toho Tech.) and an X-Y recorder (Graphtech). Cyclic voltammetry at higher potential sweep rates and double potential-step chronoamperometry were performed with a computercontrolled electrochemical measurement system (CS1090, Cypress Systems). A rotating disk electrode system (Nikko Keisoku) was employed for hydrodynamic voltammetry. Trace amounts of water in acetonitrile solutions were measured by an AQ-7 trace water measurement system (Hiranuma) based on a Karl Fischer coulometric titration. Solutions in the electrochemical cell were deoxygenated with N2 gas which was passed through concentrated sulfonic acid and silica gel traps to eliminate trace water and then an acetonitrile trap. All electrochemical measurements were performed in AN solutions containing 0.1 M (1 M= 1 mol dm − 3) TEAP under a N2 gas atmosphere at room temperature (239 2°C).
3. Results and discussion
3.1. Cyclic 6oltammetry of MCLA and its conjugate base
Scheme 1.
Fig. 1 shows representative cyclic voltammograms of 0.5 mM MCLA in AN solution containing 0.1 M
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T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
Fig. 1. Typical cyclic voltammograms of 0.50 mM MCLA obtained at a BPG electrode in AN solution containing 0.1 M TEAP. Potential scan rate: (A) 5, (B) 50, and (C) 200 mV s − 1.
TEAP at different scan rates (6). Two oxidation peaks (I ap1, I ap2) were obtained at about +0.6 and + 0.8 V during the first forward scan. The amplitude of the corresponding reduction peaks (I cp1, I cp2) on scan reversal depended on 6 and the ratios of I cp1 to I ap1 (and I cp2 to I ap2) became larger at larger scan rates. In this case, the third reduction peak (I cp3) was observed as a broad one around 0 V. This peak was not obtained when the potential scan was reversed prior to the second oxidation step. Those facts suggest that some chemical reactions follow each of the electrode reactions at +0.6 and + 0.8 V, which will be designated conveniently as Processes I and II, respectively. Fig. 2 shows typical cyclic voltammogram obtained when 2,6-lutidine (2,6-L) was added into the AN solution containing 0.1 M TEAP and 0.36 mM MCLA. 2,6-L is electroinactive in the range of potential from −0.4 to +0.9 V. As can be seen from the comparison
Fig. 2. Cyclic voltammogram of 0.36 mM MCLA obtained at a BPG electrode in AN solutions containing 0.1 M TEAP and 72.5 mM 2,6-lutidine. Potential scan rate was 50 mV s − 1.
of Fig. 1B and Fig. 2, when 2,6-L was added into the AN solution, I ap1 decreased and the new oxidation peak current (I ap1%) appeared at a more negative potential than E ap1 which corresponds to I ap1. This suggests that Process I is an electrode reaction coupled with a proton transfer. I ap1% gradually became large and the corresponding peak potential (E ap1%) shifted to the direction of negative potential, as the concentration of 2,6-L added was increased. This new electrode process in the solution containing a large excess of 2,6-L will be designated as Process I%. Processes I and II are quite close to each other. On the other hand, Process I% is sufficiently separated from Process II. The presence of base in solutions allows us to investigate easily and quantitatively the respective electrode reactions in Processes I% and II, and their coupled chemical reactions. From the above results together with the previous data [5,6] concerning the chemical redox behavior of luciferin, the electrode reaction of MCLA in solutions containing excess proton acceptors may be represented schematically by Scheme 2A, where MCLA− and MCLAOH are a conjugate base and a hydroxylated form (a luciferinol derivative) of MCLA−, respectively, and MCLA and MCLA+ are one-electron and twoelectron oxidized forms of MCLA−, respectively, and (MCLA)2 is a dimer of MCLA.
3.2. Determination of the diffusion coefficient and the number of electrons in6ol6ed in the electrode reaction of the conjugate base of MCLA MCLA− suffers a two-step oxidation in the range of potentials from − 0.4 to + 0.9 V. The number of electrons (n) involved in Process I% was determined by hydrodynamic voltammetry using a rotating disk electrode [16]. Fig. 3A shows a typical hydrodynamic voltammogram recorded for the oxidation of 0.30 mM MCLA in AN solutions containing 48 mM 2,6-L. In the presence of such an excess of 2,6-L, a sufficient separation of the two oxidation waves enabled us to determine the value of n for Process I%. Fig. 3B shows a logarithmic plot for the oxidation of Process I%. From the slope (17.691.1 V − 1), we obtained n= 1.09 0.1. The diffusion coefficient (D) of MCLA− was estimated to be (8.69 1.0)× 10 − 6 cm2 s − 1 from the slope of the Levich plot shown in Fig. 3C1. Thus it was found that Process 1 In a similar way, the values of D and n for process I were also determined in the absence of 2,6-L. However, the values obtained contained large errors because of the relatively small wave-separation on voltammograms for Processes I and II. The electrode reaction of Process I was irreversible because the slope of the logarithmic plot was 9.5 9 1.2 V − 1 which was much smaller than that (16.9 V − 1) expected for a reversible system, and so with the assumption of a (transfer coefficient) = 0.5, we obtained n =1.1 90.2. The diffusion coefficient of MCLA was estimated to be (2.3 90.5) ×10 − 5 cm2 s − 1.
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
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metric dimer having an ethyl group, they found that asymmetric dimers were produced, and that three kinds of dimers (i.e. diethyl-, dimethyl- and ethyl, methyldimers) soon coexisted at equilibrium. Thus they concluded that a reversible dimerization of radical species generated by one-electron oxidation of luciferin and its analogues took place. Fujimori et al. [8] also have proposed that the oxidation of CLA brings about the similar reversible dimerization, based on the cyclic voltammograms obtained for the oxidation of CLA in AN solutions. A similar dimerization may be expected also for the electro-oxidation of MCLA− and Process I% followed by the reversible dimerization is represented schematically as follows: MCLA− − e− MCLA kf( = Kkb)
2MCLA
X k b
(MCLA)2
(1) (2)
where kf is the forward rate constant of the dimerization, kb is the backward rate constant and K is the equilibrium constant. In the presence of an excess of 2,6-L, the electrode reaction of the MCLA−/MCLA couple is reversible as mentioned in the preceding section. Thus, the electrode reaction of MCLA− is designated as an ‘ErCr mechanism’, i.e. a reversible electrode reaction (Er) is followed by a reversible chemical reaction (Cr). This could be confirmed by the cyclic voltam-
Scheme 2.
I% was a one-electron transfer reaction. As shown in Scheme 1, two-electron oxidation of Cypridina luciferin generates Cypridina luciferinol. Therefore Process II should also be a one-electron oxidation process. However, the value of n involved in Process II could not be estimated in the same manner as mentioned above. In this case, Process II is affected by the follow-up chemical reaction of Process I% (i.e. dimerization of MCLA).
3.3. Dimerization According to Scheme 1, Cypridina luciferin (L) generates its dimers via one-electron oxidation with oxidizing reagents, the dimer and the corresponding monomer (L) are at equilibrium and L can be reduced to L with reducing reagents. Goto et al. [6] identified the dimers produced by the one-electron oxidation of luciferin and its analogues. When a dichloromethane solution of the analogous symmetric dimer having a methyl group was mixed with a dichloromethane solution of another sym-
Fig. 3. (A) Typical steady-state current-potential curve obtained with a rotating disk GC electrode in AN solution containing 0.30 mM MCLA, 0.1 M TEAP and 48 mM 2,6-lutidine. Electrode rotation rate: 400 rpm. Potential scan rate: 5 mV s − 1. (B) Log-plot for the oxidation of Process I%. (C) Levich plot of limiting current versus (electrode rotation rate)1/2 for the oxidation of Process I%.
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T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
Fig. 5. Typical DPSCA plot of ( − I c(t =2t)/I a(t =t)) versus log(tcMCLA−). cMCLA− =0.49 mM. Dashed curve indicates the simulated ( −I c(t =2t)/I a(t =t)) versus log (kftcMCLA−) curve for the radical-radical dimerization of MCLA with kf =4.1 × 104 M − 1 s − 1 and kb =3.2 × 101 s − 1.
Fig. 4. (A) Potential scan rate dependence of cyclic voltammograms of 0.50 mM MCLA in AN solution containing 0.1 M TEAP and 72 mM 2,6-lutidine. Potential scan rate: (1) 200, (2) 50 and (3) 5 mV s − 1. Current scale, S: (1) 10; (2) 5 and (3) 2 mA. (B) Potential scan rate dependence of the peak current ratio, ( − I %p1c/I %p1a).
mograms obtained for the one-electron redox reaction of 0.50 mM MCLA in AN solutions containing 72 mM 2,6-L (Fig. 4): The ratio of the reduction peak current (I cp1%) to the oxidation peak current (I ap1%) decreased with increasing potential scan rate (Fig. 4B), being consistent with the expectation for the ErCr case [17 – 20]. In order to investigate quantitatively the reversible dimerization reaction under consideration, we used double potential-step chronoamperometry (DPSCA) [21 – 23]. The starting electrode potential (E1: − 0.4 V vs. Ag AgCl NaCl(sat)) was chosen so that no reaction occurs, then the potential was stepped to a potential (E2: + 0.4), where the oxidation of a conjugate base of MCLA is diffusion-controlled. After the electrode potential was held for a time (t) at the E2 it was stepped back to the E1 again, where the reduction of the electrogenerated radical species, MCLA, is diffusioncontrolled. Thus the ratios of the reduction current (I c) at t = 2t to the oxidation current (I a) at t =t, ( −I c(t= 2t)/I a(t=t)), were measured for various values of t in the range of 50–5000 ms. The results obtained were analyzed according to the working curves of DPSCA for an ErCr (a radical – radical dimerization) case, which were obtained by Hanafey et al. [21] using a digital simulation. Fig. 5 shows the typical plot of (− I c(t= 2t)/I a(t=t)) versus log (tcMCLA−). The theoretical
(− I c(t=2t)/I a(t=t))−log (kftcMCLA−) curve with which the experimental points were matched satisfactorily is also shown in Fig. 5. From the comparison of these experimental and theoretical curves, the kinetic parameters for the radical–radical (MCLA –MCLA) dimerization (kf, kb and K (=kf/kb)) were estimated and are summarized in Table 1. Here it should be noted that in general, another mechanism is also possible for the dimerization under consideration, i.e. a parent–radical (MCLA− –MCLA) dimerization. According to the results of simulation by Hanafey et al. [21], current ratios for this case are expected to be less than 0.3 for any values of log (kftcMCLA−). The values actually obtained are more than 0.3 (see Fig. 5). Thus, this possibility was ruled out.
3.4. Hydroxylation Fig. 6 shows typical cyclic voltammograms of 0.50 mM MCLA in the AN solutions containing 72 mM 2,6-L and various concentrations of water. The redox responses observed at ca. 0 and 0.6 V correspond to Process I% and Process II, respectively, as mentioned above. We can see that the peak current ratio for Table 1 Kinetic parameters for reversible dimerization of MCLA radicals electrogenerated by one-electron oxidation of MCLA− in acetonitrile solutions in the presence of an excess of 2,6-L (72 mM) cMCLA−/mM 10−4 kf/M−1 s−1 10−1 kb/s−1 0.49 0.24
4.1 9 0.9 5.4 9 1.2 Av. 4.9 9 1.7
3.2 9 0.7 4.2 9 1.0 Av. 3.9 91.4
10−3 K/M−1 1.4 9 0.6 1.4 9 0.5 Av. 1.4 90.6
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
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the values of − I cp2/I ap2 were almost close to unity. The E1/2 value was thus estimated to be + 576930 mV versus Ag AgCl NaCl(sat). Under the present experimental conditions, the comproportionation reaction of MCLA− and MCLA+ to form MCLA could also occur: MCLA− + MCLA+
Fig. 6. Cyclic voltammograms of 0.50 mM MCLA obtained at a BPG electrode in AN solutions containing 0.1 M TEAP, 72 mM 2, 6-lutidine and various concentrations of water. cH2O: (1) 0.06; (2) 0.12 and (3) 0.87 M. Potential scan rate: 500 mV s − 1.
Process II, (− I cp2/I ap2), clearly depends on the concentration of water (cH2O), i.e. it decreases with increasing cH2O. Results of cyclic voltammetric measurements, together with Scheme 1, suggest that the rate of the follow-up chemical reaction coupled with Process II is enhanced by the addition of water and that the followup reaction is the hydroxylation of a carbocation species (MCLA+) generated by two-electron oxidation of MCLA−. In this case, the electrode reaction is represented schematically as follows: MCLA −e− MCLA+ kH O 2
MCLA+ +H2O MCLA −OH + H+
(3)
Comproportionation
_`
Disproportionation
2MCLA
(5)
The equilibrium constant of comproportionation [24,25], Keq = exp{(F/RT)DE°%} # 1.5× 109, can be calculated from the difference, DE°%, in the formal potentials of the first (E°%= + 0.0339 0.023 V for the MCLA−/MCLA couple) and second (E°% = +0.5769 0.030 V for the MCLA/MCLA+ couple) electrontransfer reactions in the presence of excess 2,6-L. Therefore, if it is assumed that the disproportionation of MCLA is actually negligible, then k%H2O is expressed as a function of cH2O and cMCLA− as follows: k%H2O = kH2OcH2O + kcompcMCLA−
(6)
where kcomp is the rate constant of the comproportionation reaction. Fig. 7B shows the values of k%H2O as a function of cH2O at cMCLA− = 0.5 mM. As expected from Eq. (6), the plot of k%H2O versus cH2O is linear with a positive intercept. Thus, kH2O and kcomp were estimated to be 8.99 1.0 M − 1 s − 1 and (1.89 0.2)× 103 M − 1 s − 1, respectively, from the slope and intercept of the plot.
3.5. Reaction mechanism The present results and previous data concerning the redox behavior of luciferin and its analogues demon-
(4)
where kH2O is the rate constant of the second-order hydroxylation reaction. In the present system, the second-order hydroxylation reaction can be treated as a pseudo-first-order reaction because H2O is in excess in comparison with MCLA− ((cH2O/cMCLA−) \20). The method of Nicholson and Shain [17] with cyclic voltammetry was applied to examine the kinetics of the hydroxylation. The rate constant (k%H2O) of the pseudo first-order hydroxylation reaction could be estimated using the working curve which is constructed for the ratio of peak currents as a function of kt% by Nicholson and Shain [17], where in the present case k corresponds to k%H2O and t% is the time in seconds from the half-wave potential E1/2 to the switching potential El, i.e. t%= E1/2 −El /n. The value of E1/2 was estimated by assuming that E1/2 # E°%# (E ap2 +E cp2)/2, where E°%, E ap2 and E cp2 are the formal redox potential, anodic and cathodic peak potentials, respectively of the cyclic voltammogram for Process II. The cyclic voltammograms for this purpose were obtained at 6= 10 – 50 V s − 1, where
Fig. 7. (A) Dependence of ( − I cp2/I ap2) on cH2O. (I cp2/I ap2)’s were estimated from cyclic voltammograms of 0.50 mM MCLA obtained at 500 mV s − 1 in AN solutions containing 0.1 M TEAP, 72 mM 2,6-lutidine and various concentrations of water. (B) Dependence of k%H2O on cH2O.
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strate that the oxidation process of MCLA− is a twostep oxidation process (see Scheme 2B). In a basic AN solution, the first oxidation step is Process I%, where a one-electron electrode reaction of MCLA− to MCLA is followed by a reversible dimerization. The radical species (MCLA) generated by the one-electron oxidation of MCLA form the dimer ((MCLA)2), and then the dissociation of (MCLA)2 to MCLA also occurs. Fujimori et al. [7] considered four resonance structures for a chemical structure of MCLA as shown in Eq. (7). The structure of 2 in Eq. (7), where an unpaired electron localizes at the 5-position, has a maximum spin concentration of radicals. Goto et al. [6] have clarified that radical species generated by one-electron chemical oxidation of Cypridina luciferin and its analogues combine reversibly at the 5-position affording the dimer. Thus the structure 2 is represented as the chemical structure of MCLA in Scheme 2B.
rate constant (kcomp) of the comproportionation of MCLA− and MCLA+, and the diffusion coefficient (DMCLA − ) of MCLA− have been estimated: kf = (4.99 1.7)× 104 M − 1 s − 1, kb = (3.99 1.4)× 101 s − 1, K= (1.49 0.6)× 103 M − 1, kH2O = 8.99 1.0 M − 1 s − 1, kcomp = (1.89 0.2)× 103 M − 1 s − 1, DMCLA− =(8.69 1.0)× 10 − 6 cm2 s − 1.
Acknowledgements This research was supported financially by Grantin-Aids for Scientific Research in Priority Areas of ‘New Polymers and Their Nano-Organized Systems’ (No. 277/0923221, 1012619) and ‘Scientific Research (A)’ (No. 10305064) from the Ministry of Education, Science, Sports and Culture, Japan, and the Katoh Science Foundation, Japan.
References (7)
The second oxidation step is Process II where a carbocation species (MCLA+) is generated by one-electron oxidation of MCLA at the electrode, and then the hydroxylation of MCLA+ and the comproportionation of MCLA− and MCLA+ take place. In the hydroxylation, the addition of a water molecule to MCLA+ and the subsequent deprotonation result in luciferinol analogue (MCLAOH).
4. Conclusions The conjugate base of MCLA, MCLA− is oxidized in two one-electron steps to the radical species MCLA and further to the carbocation species MCLA+ in AN solutions. The radical – radical coupling of MCLA occurs to form the dimer (MCLA)2 which is in equilibrium with MCLA. The addition reaction of H2O to MCLA+ produces the hydroxylated form MCLAOH of luciferinol type. The proposed mechanism (Scheme 2B) of the electrochemical redox reactions of MCLA− is essentially the same as that (Scheme 1) proposed previously for the chemical redox reactions of luciferin and its analogues, although the reduction of MCLAOH remains unclear. The rate constants (kf, kb) and equilibrium constant (K) of the reversible dimerization of MCLA, the rate constant (kH2O) of the hydroxylation of MCLA+, the
[1] Y. Toya, T. Kayano, K. Sato, T. Goto, Bull. Chem. Soc. Jpn. 65 (1992) 2475. [2] T. Goto, Pure and Appl. Chem. 17 (1968) 421. [3] F. McCapra, Y.C. Chang, J. Chem. Soc., Chem. Commun. (1967)1011. [4] T. Goto, in: W. Ando, Y. Moro-oka (Eds.), The Role of Oxygen in Chemistry and Biochemistry, Proceedings of an International Symposium on Activation of Dioxygen and Homogeneous Catalytic Oxidations, Tukuba, Japan, 12 –16 July 1987, Studies in Organic Chemistry, Elsevier, Amsterdam, 1988, p. 367. [5] Y. Toya, S. Nakatsuka, T. Goto, Tetrahedron Lett. (1983) 5753. [6] Y. Toya, S. Nakatsuka, T. Goto, Tetrahedron Lett. (1985) 239. [7] K. Fujimori, H. Nakajima, K. Akutsu, M. Mitani, H. Sawada, M. Nakayama, J. Chem. Soc. Perkin Trans. 2 (1993) 2405. [8] K. Akutsu, H. Nakajima, T. Katoh, S. Kino, K. Fujimori, J. Chem. Soc. Perkin Trans. 2 (1995) 1699. [9] T. Okajima, K. Tokuda, T. Ohsaka, Bioelectrochem. Bioenerg. 41 (1996) 205. [10] T. Okajima, K. Tokuda, T. Ohsaka, Denki Kagaku (presently Electrochemistry) 64 (1996) 1276. [11] M. Nakano, K. Sugioka, Y. Ushijima, T. Goto, Anal. Biochem. 159 (1986) 363. [12] A. Nishida, H. Kimura, M. Nakano, T. Goto, Clinica Chimica Acta 179 (1989) 177. [13] M. Nakano, Methods in Enzymology 186 (1990) 585. [14] M. Tsushima, K. Tokuda, T. Ohsaka, Anal. Chem. 66 (1994) 4551. [15] T. Ohsaka, M. Tsushima, K. Tokuda, Bioelectrochem. Bioenerg. 31 (1993) 289. [16] A.J. Bard, L.R. Faulkner (Eds.), Electrochemical Methods, Fundamentals and Applications, Wiley, New York, 1980. [17] R.S. Nicholson, I. Shain, Anal. Chem. 36 (1964) 706. [18] J.M. Saveant, E. Vianello, Electrochim. Acta 12 (1967) 1545. [19] C.P. Andrieux, L. Nadjo, J.M. Saveant, J. Electroanal. Chem. 26 (1970) 147.
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53 [20] D.H. Evans, P.J. Jimenez, M.J. Kelly, J. Electroanal. Chem. 163 (1984) 145. [21] M.K. Hanafey, R.L. Scott, T.H. Ridgway, C.N. Reilley, Anal. Chem. 50 (1978) 116. [22] C. Amatore, J. Pinson, J.M. Saveant, J. Electroanal. Chem. 137
53
(1982) 143. [23] C. Amatore, D. Garreau, M. Hammi, J. Pinson, J.M. Saveant, J. Electroanal. Chem. 184 (1985) 1. [24] C.L. Bird, A.T. Kuhn, Chem. Soc. Rev. 10 (1981) 49. [25] D.E. Richardson, H. Taube, Inorg. Chem. 20 (1981) 1278.
.
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Electrochemical behavior of a Cypridina luciferin analogue in acetonitrile solutions in the presence of proton acceptors Takeyoshi Okajima, Koichi Tokuda, Takeo Ohsaka * Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226 -8502, Japan Received 2 March 1999; received in revised form 22 June 1999; accepted 10 August 1999
Abstract The electrochemical redox behavior of the Cypridina luciferin analogue, 6-(4-methoxyphenyl)-2-methylimidazo[1, 2-a]pyrazin3(7H)-one (MCLA) has been examined in acetonitrile solutions containing 2,6-lutidine or water using cyclic and hydrodynamic voltammetry and double potential-step chronoamperometry. The conjugate base of MCLA (MCLA−) is oxidized in two one-electron steps to the radical species (MCLA) and further to the carbocation species (MCLA+). The radical – radical coupling of MCLA yields the corresponding dimer ((MCLA)2), which is in equilibrium with MCLA. MCLA+ undergoes the addition of a water molecule to yield a hydroxylated form (MCLAOH) of luciferinol type. The whole mechanism of the electrochemical redox reactions of MCLA− coupled with these chemical reactions has been found to be essentially the same as that proposed for the chemical redox reactions of Cypridina luciferin and its analogues, and in addition the kinetic data concerning the dimerization of MCLA, the dissociation of (MCLA)2 and the hydroxylation of MCLA+ have been estimated together with the diffusion coefficient of MCLA−. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Cypridina luciferin analogues; Redox reactions; Dimerization; Hydroxylation; Cyclic voltammetry; Double potential-step chronoamperometry; Hydrodynamic voltammetry
1. Introduction The present paper presents the electrochemical behavior of 6-(4-methoxyphenyl)-2-methylimidazo[1,2a]pyrazin-3(7H)-one (MCLA), which is one of the analogues of Cypridina hilgendorfii luciferin [1], in acetonitrile media.
Cypridina luciferin reacts with molecular oxygen (3O2), and consequently emits light in the presence of Cypridina luciferase [2–4]. This system is of great interest, because it is the simplest system of the enzymatic * Corresponding author. Tel.: +81-45-924-5404; fax: + 81-45-9245489. E-mail address: [email protected] (T. Ohsaka)
reactions found in bioluminescence. Goto et al. isolated Cypridina luciferin in a crystalline state and elucidated its structure completely [2]. They also prepared some analogues of luciferin and examined their chemiluminescence properties. In order to clarify the reaction mechanisms of autoxidation of luciferin in the absence of the luciferase, Goto and co-workers investigated the chemical redox reactions of Cypridina luciferin and its analogues by use of oxidizing agents ([Fe(CN)6]3 − , PbO2 and DPPH (2,2-diphenyl-1-picryl hydrazyl)) and reducing agents (Na2S2O4 and NaBH4) in dimethyl sulfoxide, water, various alcohols and water+ alcohol mixtures in the absence of Cypridina luciferase [5,6]. According to the mechanism proposed by them (Scheme 1), the one-electron oxidation of luciferin (L) gives a radical species (L), which is in equilibrium with its dimer species, biluciferyl (LL). L is reduced reversibly with Na2S2O4 to L. L is further oxidized with DPPH to luciferinol and its derivatives (LOR), which can be reduced with Na2S2O4 (or NaBH4) to L. LOR is also generated by two-electron oxidation of L with
0022-0728/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 9 9 ) 0 0 3 6 2 - 9
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PbO2. No quantitative kinetic investigation on the redox and chemical reactions of luciferin has been reported. Recently, the mechanism and kinetics of autoxidation of its analogue, 2-methyl-6-phenylimidazo[1,2-a]pyrazin3(7H)-one (CLA) have been examined, based on the chemiluminescence measurements, in aqueous solutions of various pHs under aerobic conditions [7,8]. The aim of the present study is to examine the mechanisms of the electrochemical redox reactions of MCLA and the ensuing chemical reactions (i.e. the dimerization of the one-electron oxidation product (MCLA), the dissociation of the dimer ((MCLA)2), and the hydroxylation of the two-electron oxidation product (MCLA+) of MCLA) in acetonitrile media and to obtain the related quantitative kinetic information. The experiments were limited to those in the presence of proton acceptors under a N2 atmosphere to avoid the complexity of the situation resulting from the electrochemical redox reactions of MCLA itself, and the chemical reactions of MCLA and its electrolysis products with molecular oxygen or its electrolytic reduction products (e.g. superoxide ion: O− 2 and H2O2) [9 – 13]. An investigation of the electrochemical behavior of the deprotonated MCLA is of interest in relation to the bioluminescence of Cypridina luciferin. In the proposed mechanisms of bioluminescence, a conjugate base of Cypridina luciferin is essential to the initial reaction. The results obtained confirmed the mechanism proposed by Goto et al. for the chemical redox reactions of luciferin, and in addition the kinetic data concerning the dimerization of MCLA, the dissociation of (MCLA)2, and the hydroxylation of MCLA+ were estimated together with the diffusion coefficient of the deprotonated species (MCLA−) as well as the number of electrons involved in its electrode reaction.
47
2. Experimental
2.1. Reagents MCLA (Tokyo Kasei Chemicals Industries) was a specially prepared analytical reagent and was used without further purification. Acetonitrile (AN) of reagent grade was obtained from Kanto Chemical and was used after drying with molecular sieves 4A1/16 (Wako Pure Chemicals Industries). Tetraethylammonium perchlorate (TEAP, Tokyo Kasei) as a supporting electrolyte, and 2,6-dimethylpyridine (2,6-lutidine) (Kanto) as a proton acceptor were used as received. Water was purified by passage through a Millipore Milli-Q purification train. Basal-plane pyrolytic graphite (BPG, Union Carbide) and glassy carbon (GC, Tokai Carbon) were used as the working electrodes. BPG (area: 1.8× 10 − 1 cm2) and GC (area: 7.9 × 10 − 3 cm2) disk electrodes were prepared and pre-treated before use as described previously [14].
2.2. Apparatus and procedures The electrochemical cell and its components were the same as reported previously [15]. BPG and GC disk electrodes were used as the working electrodes, a spiral platinum wire as the auxiliary electrode, and a sodium chloride saturated silver silver chloride electrode (Ag AgCl NaCl(sat)) and Ag wire as the reference electrodes. Cyclic voltammetry in the range of potential sweep rates from 2 to 500 mV s − 1 was carried out using a PS-07 polarization unit (Toho Tech.) and an X-Y recorder (Graphtech). Cyclic voltammetry at higher potential sweep rates and double potential-step chronoamperometry were performed with a computercontrolled electrochemical measurement system (CS1090, Cypress Systems). A rotating disk electrode system (Nikko Keisoku) was employed for hydrodynamic voltammetry. Trace amounts of water in acetonitrile solutions were measured by an AQ-7 trace water measurement system (Hiranuma) based on a Karl Fischer coulometric titration. Solutions in the electrochemical cell were deoxygenated with N2 gas which was passed through concentrated sulfonic acid and silica gel traps to eliminate trace water and then an acetonitrile trap. All electrochemical measurements were performed in AN solutions containing 0.1 M (1 M= 1 mol dm − 3) TEAP under a N2 gas atmosphere at room temperature (239 2°C).
3. Results and discussion
3.1. Cyclic 6oltammetry of MCLA and its conjugate base
Scheme 1.
Fig. 1 shows representative cyclic voltammograms of 0.5 mM MCLA in AN solution containing 0.1 M
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T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
Fig. 1. Typical cyclic voltammograms of 0.50 mM MCLA obtained at a BPG electrode in AN solution containing 0.1 M TEAP. Potential scan rate: (A) 5, (B) 50, and (C) 200 mV s − 1.
TEAP at different scan rates (6). Two oxidation peaks (I ap1, I ap2) were obtained at about +0.6 and + 0.8 V during the first forward scan. The amplitude of the corresponding reduction peaks (I cp1, I cp2) on scan reversal depended on 6 and the ratios of I cp1 to I ap1 (and I cp2 to I ap2) became larger at larger scan rates. In this case, the third reduction peak (I cp3) was observed as a broad one around 0 V. This peak was not obtained when the potential scan was reversed prior to the second oxidation step. Those facts suggest that some chemical reactions follow each of the electrode reactions at +0.6 and + 0.8 V, which will be designated conveniently as Processes I and II, respectively. Fig. 2 shows typical cyclic voltammogram obtained when 2,6-lutidine (2,6-L) was added into the AN solution containing 0.1 M TEAP and 0.36 mM MCLA. 2,6-L is electroinactive in the range of potential from −0.4 to +0.9 V. As can be seen from the comparison
Fig. 2. Cyclic voltammogram of 0.36 mM MCLA obtained at a BPG electrode in AN solutions containing 0.1 M TEAP and 72.5 mM 2,6-lutidine. Potential scan rate was 50 mV s − 1.
of Fig. 1B and Fig. 2, when 2,6-L was added into the AN solution, I ap1 decreased and the new oxidation peak current (I ap1%) appeared at a more negative potential than E ap1 which corresponds to I ap1. This suggests that Process I is an electrode reaction coupled with a proton transfer. I ap1% gradually became large and the corresponding peak potential (E ap1%) shifted to the direction of negative potential, as the concentration of 2,6-L added was increased. This new electrode process in the solution containing a large excess of 2,6-L will be designated as Process I%. Processes I and II are quite close to each other. On the other hand, Process I% is sufficiently separated from Process II. The presence of base in solutions allows us to investigate easily and quantitatively the respective electrode reactions in Processes I% and II, and their coupled chemical reactions. From the above results together with the previous data [5,6] concerning the chemical redox behavior of luciferin, the electrode reaction of MCLA in solutions containing excess proton acceptors may be represented schematically by Scheme 2A, where MCLA− and MCLAOH are a conjugate base and a hydroxylated form (a luciferinol derivative) of MCLA−, respectively, and MCLA and MCLA+ are one-electron and twoelectron oxidized forms of MCLA−, respectively, and (MCLA)2 is a dimer of MCLA.
3.2. Determination of the diffusion coefficient and the number of electrons in6ol6ed in the electrode reaction of the conjugate base of MCLA MCLA− suffers a two-step oxidation in the range of potentials from − 0.4 to + 0.9 V. The number of electrons (n) involved in Process I% was determined by hydrodynamic voltammetry using a rotating disk electrode [16]. Fig. 3A shows a typical hydrodynamic voltammogram recorded for the oxidation of 0.30 mM MCLA in AN solutions containing 48 mM 2,6-L. In the presence of such an excess of 2,6-L, a sufficient separation of the two oxidation waves enabled us to determine the value of n for Process I%. Fig. 3B shows a logarithmic plot for the oxidation of Process I%. From the slope (17.691.1 V − 1), we obtained n= 1.09 0.1. The diffusion coefficient (D) of MCLA− was estimated to be (8.69 1.0)× 10 − 6 cm2 s − 1 from the slope of the Levich plot shown in Fig. 3C1. Thus it was found that Process 1 In a similar way, the values of D and n for process I were also determined in the absence of 2,6-L. However, the values obtained contained large errors because of the relatively small wave-separation on voltammograms for Processes I and II. The electrode reaction of Process I was irreversible because the slope of the logarithmic plot was 9.5 9 1.2 V − 1 which was much smaller than that (16.9 V − 1) expected for a reversible system, and so with the assumption of a (transfer coefficient) = 0.5, we obtained n =1.1 90.2. The diffusion coefficient of MCLA was estimated to be (2.3 90.5) ×10 − 5 cm2 s − 1.
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
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metric dimer having an ethyl group, they found that asymmetric dimers were produced, and that three kinds of dimers (i.e. diethyl-, dimethyl- and ethyl, methyldimers) soon coexisted at equilibrium. Thus they concluded that a reversible dimerization of radical species generated by one-electron oxidation of luciferin and its analogues took place. Fujimori et al. [8] also have proposed that the oxidation of CLA brings about the similar reversible dimerization, based on the cyclic voltammograms obtained for the oxidation of CLA in AN solutions. A similar dimerization may be expected also for the electro-oxidation of MCLA− and Process I% followed by the reversible dimerization is represented schematically as follows: MCLA− − e− MCLA kf( = Kkb)
2MCLA
X k b
(MCLA)2
(1) (2)
where kf is the forward rate constant of the dimerization, kb is the backward rate constant and K is the equilibrium constant. In the presence of an excess of 2,6-L, the electrode reaction of the MCLA−/MCLA couple is reversible as mentioned in the preceding section. Thus, the electrode reaction of MCLA− is designated as an ‘ErCr mechanism’, i.e. a reversible electrode reaction (Er) is followed by a reversible chemical reaction (Cr). This could be confirmed by the cyclic voltam-
Scheme 2.
I% was a one-electron transfer reaction. As shown in Scheme 1, two-electron oxidation of Cypridina luciferin generates Cypridina luciferinol. Therefore Process II should also be a one-electron oxidation process. However, the value of n involved in Process II could not be estimated in the same manner as mentioned above. In this case, Process II is affected by the follow-up chemical reaction of Process I% (i.e. dimerization of MCLA).
3.3. Dimerization According to Scheme 1, Cypridina luciferin (L) generates its dimers via one-electron oxidation with oxidizing reagents, the dimer and the corresponding monomer (L) are at equilibrium and L can be reduced to L with reducing reagents. Goto et al. [6] identified the dimers produced by the one-electron oxidation of luciferin and its analogues. When a dichloromethane solution of the analogous symmetric dimer having a methyl group was mixed with a dichloromethane solution of another sym-
Fig. 3. (A) Typical steady-state current-potential curve obtained with a rotating disk GC electrode in AN solution containing 0.30 mM MCLA, 0.1 M TEAP and 48 mM 2,6-lutidine. Electrode rotation rate: 400 rpm. Potential scan rate: 5 mV s − 1. (B) Log-plot for the oxidation of Process I%. (C) Levich plot of limiting current versus (electrode rotation rate)1/2 for the oxidation of Process I%.
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T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
Fig. 5. Typical DPSCA plot of ( − I c(t =2t)/I a(t =t)) versus log(tcMCLA−). cMCLA− =0.49 mM. Dashed curve indicates the simulated ( −I c(t =2t)/I a(t =t)) versus log (kftcMCLA−) curve for the radical-radical dimerization of MCLA with kf =4.1 × 104 M − 1 s − 1 and kb =3.2 × 101 s − 1.
Fig. 4. (A) Potential scan rate dependence of cyclic voltammograms of 0.50 mM MCLA in AN solution containing 0.1 M TEAP and 72 mM 2,6-lutidine. Potential scan rate: (1) 200, (2) 50 and (3) 5 mV s − 1. Current scale, S: (1) 10; (2) 5 and (3) 2 mA. (B) Potential scan rate dependence of the peak current ratio, ( − I %p1c/I %p1a).
mograms obtained for the one-electron redox reaction of 0.50 mM MCLA in AN solutions containing 72 mM 2,6-L (Fig. 4): The ratio of the reduction peak current (I cp1%) to the oxidation peak current (I ap1%) decreased with increasing potential scan rate (Fig. 4B), being consistent with the expectation for the ErCr case [17 – 20]. In order to investigate quantitatively the reversible dimerization reaction under consideration, we used double potential-step chronoamperometry (DPSCA) [21 – 23]. The starting electrode potential (E1: − 0.4 V vs. Ag AgCl NaCl(sat)) was chosen so that no reaction occurs, then the potential was stepped to a potential (E2: + 0.4), where the oxidation of a conjugate base of MCLA is diffusion-controlled. After the electrode potential was held for a time (t) at the E2 it was stepped back to the E1 again, where the reduction of the electrogenerated radical species, MCLA, is diffusioncontrolled. Thus the ratios of the reduction current (I c) at t = 2t to the oxidation current (I a) at t =t, ( −I c(t= 2t)/I a(t=t)), were measured for various values of t in the range of 50–5000 ms. The results obtained were analyzed according to the working curves of DPSCA for an ErCr (a radical – radical dimerization) case, which were obtained by Hanafey et al. [21] using a digital simulation. Fig. 5 shows the typical plot of (− I c(t= 2t)/I a(t=t)) versus log (tcMCLA−). The theoretical
(− I c(t=2t)/I a(t=t))−log (kftcMCLA−) curve with which the experimental points were matched satisfactorily is also shown in Fig. 5. From the comparison of these experimental and theoretical curves, the kinetic parameters for the radical–radical (MCLA –MCLA) dimerization (kf, kb and K (=kf/kb)) were estimated and are summarized in Table 1. Here it should be noted that in general, another mechanism is also possible for the dimerization under consideration, i.e. a parent–radical (MCLA− –MCLA) dimerization. According to the results of simulation by Hanafey et al. [21], current ratios for this case are expected to be less than 0.3 for any values of log (kftcMCLA−). The values actually obtained are more than 0.3 (see Fig. 5). Thus, this possibility was ruled out.
3.4. Hydroxylation Fig. 6 shows typical cyclic voltammograms of 0.50 mM MCLA in the AN solutions containing 72 mM 2,6-L and various concentrations of water. The redox responses observed at ca. 0 and 0.6 V correspond to Process I% and Process II, respectively, as mentioned above. We can see that the peak current ratio for Table 1 Kinetic parameters for reversible dimerization of MCLA radicals electrogenerated by one-electron oxidation of MCLA− in acetonitrile solutions in the presence of an excess of 2,6-L (72 mM) cMCLA−/mM 10−4 kf/M−1 s−1 10−1 kb/s−1 0.49 0.24
4.1 9 0.9 5.4 9 1.2 Av. 4.9 9 1.7
3.2 9 0.7 4.2 9 1.0 Av. 3.9 91.4
10−3 K/M−1 1.4 9 0.6 1.4 9 0.5 Av. 1.4 90.6
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53
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the values of − I cp2/I ap2 were almost close to unity. The E1/2 value was thus estimated to be + 576930 mV versus Ag AgCl NaCl(sat). Under the present experimental conditions, the comproportionation reaction of MCLA− and MCLA+ to form MCLA could also occur: MCLA− + MCLA+
Fig. 6. Cyclic voltammograms of 0.50 mM MCLA obtained at a BPG electrode in AN solutions containing 0.1 M TEAP, 72 mM 2, 6-lutidine and various concentrations of water. cH2O: (1) 0.06; (2) 0.12 and (3) 0.87 M. Potential scan rate: 500 mV s − 1.
Process II, (− I cp2/I ap2), clearly depends on the concentration of water (cH2O), i.e. it decreases with increasing cH2O. Results of cyclic voltammetric measurements, together with Scheme 1, suggest that the rate of the follow-up chemical reaction coupled with Process II is enhanced by the addition of water and that the followup reaction is the hydroxylation of a carbocation species (MCLA+) generated by two-electron oxidation of MCLA−. In this case, the electrode reaction is represented schematically as follows: MCLA −e− MCLA+ kH O 2
MCLA+ +H2O MCLA −OH + H+
(3)
Comproportionation
_`
Disproportionation
2MCLA
(5)
The equilibrium constant of comproportionation [24,25], Keq = exp{(F/RT)DE°%} # 1.5× 109, can be calculated from the difference, DE°%, in the formal potentials of the first (E°%= + 0.0339 0.023 V for the MCLA−/MCLA couple) and second (E°% = +0.5769 0.030 V for the MCLA/MCLA+ couple) electrontransfer reactions in the presence of excess 2,6-L. Therefore, if it is assumed that the disproportionation of MCLA is actually negligible, then k%H2O is expressed as a function of cH2O and cMCLA− as follows: k%H2O = kH2OcH2O + kcompcMCLA−
(6)
where kcomp is the rate constant of the comproportionation reaction. Fig. 7B shows the values of k%H2O as a function of cH2O at cMCLA− = 0.5 mM. As expected from Eq. (6), the plot of k%H2O versus cH2O is linear with a positive intercept. Thus, kH2O and kcomp were estimated to be 8.99 1.0 M − 1 s − 1 and (1.89 0.2)× 103 M − 1 s − 1, respectively, from the slope and intercept of the plot.
3.5. Reaction mechanism The present results and previous data concerning the redox behavior of luciferin and its analogues demon-
(4)
where kH2O is the rate constant of the second-order hydroxylation reaction. In the present system, the second-order hydroxylation reaction can be treated as a pseudo-first-order reaction because H2O is in excess in comparison with MCLA− ((cH2O/cMCLA−) \20). The method of Nicholson and Shain [17] with cyclic voltammetry was applied to examine the kinetics of the hydroxylation. The rate constant (k%H2O) of the pseudo first-order hydroxylation reaction could be estimated using the working curve which is constructed for the ratio of peak currents as a function of kt% by Nicholson and Shain [17], where in the present case k corresponds to k%H2O and t% is the time in seconds from the half-wave potential E1/2 to the switching potential El, i.e. t%= E1/2 −El /n. The value of E1/2 was estimated by assuming that E1/2 # E°%# (E ap2 +E cp2)/2, where E°%, E ap2 and E cp2 are the formal redox potential, anodic and cathodic peak potentials, respectively of the cyclic voltammogram for Process II. The cyclic voltammograms for this purpose were obtained at 6= 10 – 50 V s − 1, where
Fig. 7. (A) Dependence of ( − I cp2/I ap2) on cH2O. (I cp2/I ap2)’s were estimated from cyclic voltammograms of 0.50 mM MCLA obtained at 500 mV s − 1 in AN solutions containing 0.1 M TEAP, 72 mM 2,6-lutidine and various concentrations of water. (B) Dependence of k%H2O on cH2O.
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strate that the oxidation process of MCLA− is a twostep oxidation process (see Scheme 2B). In a basic AN solution, the first oxidation step is Process I%, where a one-electron electrode reaction of MCLA− to MCLA is followed by a reversible dimerization. The radical species (MCLA) generated by the one-electron oxidation of MCLA form the dimer ((MCLA)2), and then the dissociation of (MCLA)2 to MCLA also occurs. Fujimori et al. [7] considered four resonance structures for a chemical structure of MCLA as shown in Eq. (7). The structure of 2 in Eq. (7), where an unpaired electron localizes at the 5-position, has a maximum spin concentration of radicals. Goto et al. [6] have clarified that radical species generated by one-electron chemical oxidation of Cypridina luciferin and its analogues combine reversibly at the 5-position affording the dimer. Thus the structure 2 is represented as the chemical structure of MCLA in Scheme 2B.
rate constant (kcomp) of the comproportionation of MCLA− and MCLA+, and the diffusion coefficient (DMCLA − ) of MCLA− have been estimated: kf = (4.99 1.7)× 104 M − 1 s − 1, kb = (3.99 1.4)× 101 s − 1, K= (1.49 0.6)× 103 M − 1, kH2O = 8.99 1.0 M − 1 s − 1, kcomp = (1.89 0.2)× 103 M − 1 s − 1, DMCLA− =(8.69 1.0)× 10 − 6 cm2 s − 1.
Acknowledgements This research was supported financially by Grantin-Aids for Scientific Research in Priority Areas of ‘New Polymers and Their Nano-Organized Systems’ (No. 277/0923221, 1012619) and ‘Scientific Research (A)’ (No. 10305064) from the Ministry of Education, Science, Sports and Culture, Japan, and the Katoh Science Foundation, Japan.
References (7)
The second oxidation step is Process II where a carbocation species (MCLA+) is generated by one-electron oxidation of MCLA at the electrode, and then the hydroxylation of MCLA+ and the comproportionation of MCLA− and MCLA+ take place. In the hydroxylation, the addition of a water molecule to MCLA+ and the subsequent deprotonation result in luciferinol analogue (MCLAOH).
4. Conclusions The conjugate base of MCLA, MCLA− is oxidized in two one-electron steps to the radical species MCLA and further to the carbocation species MCLA+ in AN solutions. The radical – radical coupling of MCLA occurs to form the dimer (MCLA)2 which is in equilibrium with MCLA. The addition reaction of H2O to MCLA+ produces the hydroxylated form MCLAOH of luciferinol type. The proposed mechanism (Scheme 2B) of the electrochemical redox reactions of MCLA− is essentially the same as that (Scheme 1) proposed previously for the chemical redox reactions of luciferin and its analogues, although the reduction of MCLAOH remains unclear. The rate constants (kf, kb) and equilibrium constant (K) of the reversible dimerization of MCLA, the rate constant (kH2O) of the hydroxylation of MCLA+, the
[1] Y. Toya, T. Kayano, K. Sato, T. Goto, Bull. Chem. Soc. Jpn. 65 (1992) 2475. [2] T. Goto, Pure and Appl. Chem. 17 (1968) 421. [3] F. McCapra, Y.C. Chang, J. Chem. Soc., Chem. Commun. (1967)1011. [4] T. Goto, in: W. Ando, Y. Moro-oka (Eds.), The Role of Oxygen in Chemistry and Biochemistry, Proceedings of an International Symposium on Activation of Dioxygen and Homogeneous Catalytic Oxidations, Tukuba, Japan, 12 –16 July 1987, Studies in Organic Chemistry, Elsevier, Amsterdam, 1988, p. 367. [5] Y. Toya, S. Nakatsuka, T. Goto, Tetrahedron Lett. (1983) 5753. [6] Y. Toya, S. Nakatsuka, T. Goto, Tetrahedron Lett. (1985) 239. [7] K. Fujimori, H. Nakajima, K. Akutsu, M. Mitani, H. Sawada, M. Nakayama, J. Chem. Soc. Perkin Trans. 2 (1993) 2405. [8] K. Akutsu, H. Nakajima, T. Katoh, S. Kino, K. Fujimori, J. Chem. Soc. Perkin Trans. 2 (1995) 1699. [9] T. Okajima, K. Tokuda, T. Ohsaka, Bioelectrochem. Bioenerg. 41 (1996) 205. [10] T. Okajima, K. Tokuda, T. Ohsaka, Denki Kagaku (presently Electrochemistry) 64 (1996) 1276. [11] M. Nakano, K. Sugioka, Y. Ushijima, T. Goto, Anal. Biochem. 159 (1986) 363. [12] A. Nishida, H. Kimura, M. Nakano, T. Goto, Clinica Chimica Acta 179 (1989) 177. [13] M. Nakano, Methods in Enzymology 186 (1990) 585. [14] M. Tsushima, K. Tokuda, T. Ohsaka, Anal. Chem. 66 (1994) 4551. [15] T. Ohsaka, M. Tsushima, K. Tokuda, Bioelectrochem. Bioenerg. 31 (1993) 289. [16] A.J. Bard, L.R. Faulkner (Eds.), Electrochemical Methods, Fundamentals and Applications, Wiley, New York, 1980. [17] R.S. Nicholson, I. Shain, Anal. Chem. 36 (1964) 706. [18] J.M. Saveant, E. Vianello, Electrochim. Acta 12 (1967) 1545. [19] C.P. Andrieux, L. Nadjo, J.M. Saveant, J. Electroanal. Chem. 26 (1970) 147.
T. Okajima et al. / Journal of Electroanalytical Chemistry 476 (1999) 46–53 [20] D.H. Evans, P.J. Jimenez, M.J. Kelly, J. Electroanal. Chem. 163 (1984) 145. [21] M.K. Hanafey, R.L. Scott, T.H. Ridgway, C.N. Reilley, Anal. Chem. 50 (1978) 116. [22] C. Amatore, J. Pinson, J.M. Saveant, J. Electroanal. Chem. 137
53
(1982) 143. [23] C. Amatore, D. Garreau, M. Hammi, J. Pinson, J.M. Saveant, J. Electroanal. Chem. 184 (1985) 1. [24] C.L. Bird, A.T. Kuhn, Chem. Soc. Rev. 10 (1981) 49. [25] D.E. Richardson, H. Taube, Inorg. Chem. 20 (1981) 1278.
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