Electrochimica Acta 56 (2011) 12191226

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Spectroelectrochemistry of solid indirubin and its sulfonated form

Xiao-Wei Hu, Jian-Bo He , Ya-Hua You, Ying-Meng Zhang, Shi-Liang Zhang
Anhui Key Lab of Controllable Chemical Reaction & Material Chemical Engineering, School of Chemical Engineering, Hefei University of Technology, Hefei 230009, China

a r t i c l e

i n f o

a b s t r a c t
Electrochemical processes of solid indirubin and its sulfonated form were comparatively studied in aqueous buffers by cyclic voltammetry and long-path-length thin-layer UVvis spectroelectrochemistry, using a carbon paste working electrode with or without indirubin particles attached. The two forms of indirubin gave similar voltammetric features as well as main reaction products. Alkaline pH of electrolyte generally had a negative effect on both the reaction systems, compared with the acidic pH. Electro-reduction of both indirubins produced their leuco forms, which can be oxidized back to the initial reactants by oxygen. In the alkaline buffers the leuco-indirubin (not sulfonated) may form aggregates with poor solubility and poor electrochemical reactivity. Electro-oxidation of both indirubins led to the irreversible formation of isatin (sulfonated or not). An EC and ECE mechanisms are proposed for the reduction and the oxidation, respectively, of indirubin in two forms. The combination of solid state and solution phase spectroelectrochemistry shows the advantage of providing multidimensional information for reaction mechanism determination. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 24 August 2010 Received in revised form 29 September 2010 Accepted 2 October 2010 Available online 2 November 2010 Keywords: UVvis spectroelectrochemistry Solid state electrochemistry Indirubin Sulfonated indirubin Carbon paste electrode

1. Introduction Indirubin, a bis-indole isomer of indigo, can be obtained from indigo-producing plants, some marine mollusks, several recombinant bacteria and mammalian urine [1]. Both indigo and indirubin were the important components of some antique dyes [24]. They and some of their derivatives are considered vat dyes, which must be reduced to their water-soluble forms (leuco form) by a strong reducing agent (such as sodium dithionite in alkaline media), prior to the dyeing process. Electrochemical reduction technologies, both direct [5] and indirect [6], have been developed mostly for indigo, as an environmental-friendly alternative to the traditional chemical reduction. Other electrochemical studies for indigo and its derivatives were aimed at their redox properties [710], electroanalysis [2,1113], and electro-oxidation for the treatment of textile wastewaters [14]. Especially, Domnech et al. have developed some electroanalytical methods for various indigoid Maya Blue systems applied to conservation and restoration of cultural heritage [2,11,1517]. Also indirubin is the active ingredient of a traditional Chinese medicine, Danggui Longhui Wan, reported as a treatment for chronic myelocytic leukemia based on its inhibitory activity on cyclin-dependent kinases [1,18,19]. Numerous indirubin deriva-

Corresponding author at: School of Chemical Engineering, Hefei University of Technology, Tunxi Road 193, Hefei, Anhui Province 230009, China. Tel.: +86 551 2901462; fax: +86 551 2901450. E-mail address: jbhe@hfut.edu.cn (J.-B. He). 0013-4686/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2010.10.008

tives have been chemically synthesized to increase the selectivity, solubility and efcacy against tumor growth [20,21]. Sulfonation of indirubin has been employed as a strategy to improve the solubility in aqueous systems [2224]. Interestingly, indigoid pigments, such as indirubin and indigo, are excreted in the urine not only of patients with various pathological conditions but also of healthy individuals and animals [18,25]. They were both present at average concentrations of 0.2 nmol dm3 in the urine of normal donors [25]. Their precursor in humans is dietary tryptophan that is subjected to a series of chemical reactions including oxidation by oxygen to form indigo and indirubin [18]. Although electrochemical studies are carried out extensively for the reduction and the oxidation of indigo, only a few reports are available for indirubin. Domnech et al. developed a voltammetryof-microparticles approach to monitor the preparation of indigo and indirubin from Indigofera suffruticosa following the procedures attributed to ancient Mayas [11]. Ye et al. reported the electrochemical and spectroscopic study for the interaction of indirubin with DNA [26]. In the present work, UVvis spectroelectrochemistry was applied to in situ monitor both the reduction and the oxidation processes of solid indirubin as well as soluble sulfonated indirubin, proceeding from a comparative strategy between solidphase and solution-phase spectroelectrochemistry. Carbon paste electrodes (CPE) with or without indirubin particles attached were used as the working electrodes assembled in a long-optical-path thin-layer electrochemical cell (LTE-cell). The CPE was selected with solid parafn wax as a binder in this work because of its advantages of low noise and background current, simply renewable surface [27,28], and especially, allowing to be bulk-modied

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Scheme 1. Chemical structures of indirubin and its isomer indigo.

with various solid powders [29]. The spectral changes shown in the soluble-reactant system reect the total results of the consumed reactant(s) and the formed product(s) coexisting in the thin-layer electrolyte solution, while those obtained from the solid-reactant system denitely originate from the formation of the product(s) that strips from the electrode surface, being helpful in identifying the electrolysis products. Therefore, the combination of solid state and solution phase spectroelectrochemistry can be expected to provide multidimensional evidences which help to gain more insight into the electrochemical mechanisms. In addition, the differences in electrochemical behavior among indirubin, sulfonated indirubin and indigo deserve careful study, since sulfonated indirubin is one of the water-soluble alternatives to indirubin in medicine eld, while indigo is a well-studied isomer of indirubin, with one different indole moiety only (Scheme 1). The spectroelectrochemistry of indigo has been recently reported by our group [10]. 2. Experimental 2.1. Chemicals and solutions Indirubin (98% purity) was purchased from Shanghai Standard Biotech Company and was used as received. Spectrograde graphite powder (320 mesh) and spectrograde parafn wax (solidication point 4648 C) were purchased from Shanghai Chemical Works for preparing the CPE. Doubly-distilled water was prepared in an all-glass distillatory apparatus for solution preparation. All other chemicals were of analytical grade from China-Reagent group. The supporting electrolytes were 0.2 mol dm3 BrittonRobinson buffered solutions (BRS) with various pH values plus 0.5 mol dm3 KCl. Sulfonated indirubin was prepared by reaction of 0.0262 g indirubin powder with 15 ml concentrated sulfuric acid at 80 C for 1 h. The sulfonated solution was neutralized by adding sodium hydroxide solution to neutral pH, and was diluted to desired concentrations with different pH BRSs prior to use. The resulting sulfonated indirubin in aqueous solution shows an UVvis spectrum similar to that of the indirubin dissolved in ethanol. Both of the spectra give ve absorption bands, among which the three bands at 535 nm, 360 nm and 290 nm show no change in wavelength upon sulfonation, but with slight changes in intensity. The other two bands below 260 nm are the E2 and B bands of benzene rings, both showing blue shift caused by the sulfonation. This can be attributed to the action of sulfonic groups, which should have been attached to the benzene rings of indirubin molecules, most probably at the 5,5 -positions [19,23,24]. 2.2. Electrode preparation A disk carbon paste working electrode with a smaller geometrical area of 6.2 mm2 was prepared for the voltammetric measurements, while a quadrate one with a larger area of 77 mm2 was fabricated for the in situ spectroelectrochemical experiments.

The disk electrode body was a polystyrene hollow tube with inner diameter of 2.8 mm, which was tightly impacted with a copper rod leaving a cavity of 2 mm depth at one end of the tube. The quadrate electrode body was a polystyrene plate with a cavity of 8 mm 9.6 mm, as described previously [30]. The solid carbon paste was made from dry graphite powder and parafn wax in a ratio of 5:2 (w/w). The wax was heated in an evaporating dish until molten, and then was mixed with the graphite powder to obtain a well-blended paste. This paste was pressed rmly into the cavities of the two kinds of electrode bodies, and then the paste surface was polished successively with 8004000 grit emery papers. The resulting CPE was washed ultrasonically in doubly-distilled water for 5 s to remove the stuck particles, and then stored in BRS prior to use. Attachment of indirubin powders onto the small disk CPE was performed by pipetting 5 l of 30 mol dm3 indirubin (dissolved in ethanol) onto the electrode surface followed by air drying. On the large quadrate CPE the attachment was done by mechanically grinding the electrode surface on indirubin powders and then polishing on the 4000 grit emery paper. The prepared indirubinattached CPEs (abbreviated to IrCPEs) were used for the solid state electrochemistry of indirubin, while the bare ones were used for hydrosoluble sulfonated indirubin. 2.3. Apparatus and procedures Electrochemical experiments including cyclic voltammetry and potential step spectroelectrochemistry were carried out on a CHI660C electrochemical analyzer (Chenhua, Shanghai, China). The working electrode was either CPE or IrCPE, used along with a KClsaturated Ag/AgCl reference electrode and a platinum coil counter electrode. A conventional single-compartment cell was used for the voltammetric measurements. The time-dependent thin-layer UVvis spectra were recorded in situ on an UV-2500 spectrophotometer (Shimadzu, Japan), to monitor the redox products of both solid indirubin and sulfonated indirubin at different potentials. The LTE-cell is home-made using a commercial available quartz photometric cell as the cell body [30], with a long-path-length of 10 mm and a thin-layer thickness of 0.2 mm. The incident monochromatic light beam was adjusted to run just along the surface of the working electrode for measuring the absorbance of the thin-layer solution. The time constant of the cell is less than 1 ms in 0.5 mol dm3 KCl solution, which was characterized by chronoamperometric experiments. Before experiment, the electrochemical cell was washed with doubly-distilled water and ethanol successively for 1 min under ultrasonication. All experiments were conducted at room temperature (22 1 C). The electrolyte solutions were not deaerated for the purpose of exploring the electrochemical behavior of indirubin in a natural environment. The bare CPEs were degreased after each run by repetitive cyclic scans between 1.2 and 1.5 V in 1.0 mol dm3 KCl waterethanol solution, until only the background current presented. The IrCPEs were used only one time, and then polished on emery papers for indirubin-reloading and the next run. 3. Results and discussion 3.1. Cyclic voltammetry 3.1.1. Reduction and re-oxidation of solid indirubin Fig. 1A presents the cyclic voltammograms (CVs) of solid indirubin at the IrCPE in different pH BRSs. A pair of current peaks were observed with a large peak-separation ( Ep ) of about 500 mV at a scan rate of 100 mV s1 (labeled as peaks I and I ), correspond-

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Fig. 2. CVs of solid indirubin at IrCPE, for investigating its oxidation. Buffer pH (1 6): 1.8, 3.4, 5.4, 7.4, 9.4, 11.4; scan rate: 100 mV s1 ; insets: CVs of 1.0 104 mol dm3 sulfonated indirubin under the corresponding conditions.

Fig. 1B shows the effect of scan rate over a range of 2.0300 mV s1 on the peak pair I and I . The cathodic-to-anodic peak potential separation increased from 440 mV to 510 mV with increasing scan rate, due to the quasi-reversibility of charge transfer and an effect of uncompensated ohmic drop. Both the peak currents mounted up with increasing scan rate, especially the reverse anodic peak I that was nearly absent at the low scan rate (2.0 mV s1 ). The ratio of anodic to cathodic peak current (Ipa /|Ipc |) increased with scan rate (inset of Fig. 1B), according with the electrochemicalchemical (EC) mechanism disposed for the solution phase electrochemistry [31]. A detailed mechanism will be given in Section 3.2.2 based on the further discussion.
Fig. 1. CVs of solid indirubin at IrCPE, for investigating its reduction and reoxidation. (A) Buffer pH (1 4): 1.8, 3.4, 5.4, 7.4; scan rate: 100 mV s1 ; inset: CVs of 1.0 104 mol dm3 sulfonated indirubin under the corresponding conditions. (B) Buffer pH 3.4; scan rate (1 8): 2, 10, 50, 100, 150, 200, 250, 300 mV s1 ; inset: the dependence of the anodic to cathodic peak current ratio on scan rate.

ing to the reduction and re-oxidation of indirubin, respectively. The large peak-separation, along with the very low reverse peak I compared with its counterpart, indicates the poor reversibility of the electro-reduction of indirubin. This feature is different remarkably from the highly reversible electrochemistry of indigo [10]. The peak I, while in acidic media, kept a symmetrical and well-dened shape as anticipated for the response arising from surface-conned species. This peak was distorted by the large blank current produced in neutral and alkaline buffers. The two peak potentials (Epc and Epa ) both exhibit linear dependence with buffer pH: Epc (I)/V = 0.1725 0.0482 pH (r = 0.9994) and Epa (I )/V = 0.3359 0.0494 pH (r = 0.9961). The slopes of both equations being about 49 mV per pH unit support a mechanism involving a near equal number of electrons and protons. A twoelectron two-proton transfer mechanism has been suggested for the reduction of indirubin to its leuco form [11], similar to that well known for indigo [7,13,15]. The reduction of indirubin was considered to occur at the C-3 and C-2 carbonyl groups forming two hydroxyl groups. These two active sites, without the structural symmetry as present in indigo, are expected to show different redox activity, but gave one cathodic peak only. A possible reaction path is that the initial charge transfer took place only at one carbonyl (showing one cathodic peak only) to form a high reactive carbon free radical, which underwent a dismutation reaction to produce the nal leuco-indirubin.

3.1.2. Oxidation of solid indirubin Direct oxidation of indirubin was observed from the anodic peak II located in the more positive potential range (Fig. 2). Absence of the corresponding cathodic peak indicates a completely irreversible oxidation process, contrary to the reversible oxidation of indigo [10]. The peak II also exhibits a linear dependence of its peak potential with pH, Epa (II)/V = 1.0699 0.04814 pH (r = 0.9949), the slope suggesting that a near equal number of electrons and protons were lost in reaction processes. The peak current of the peak II decreased with increasing pH. Furthermore, this peak does not show the symmetry in shape expected for a surface-conned species, but with a seeming diffusion tail. This tail probably resulted from subsequent oxidation step following the initial charge transfer at the peak. A multi-step oxidation mechanism will be given in Section 3.3.2. 3.1.3. Sulfonated indirubin Similar voltammetric responses, including the pH effects on both peak potentials and peak currents, were observed in the CVs of the hydrosoluble sulfonated indirubin (insets in Figs. 1A and 2). This similarity supports that the sulfonation taking place at the phenyl rings did not change the chemical structure of the electrochemically active moiety (pyrrolidinones) of indirubin. One mentionable difference is that all the three peaks (I, I and II) of the sulfonated indirubin occurred at more positive potentials than did the corresponding peaks of solid indirubin, especially the peak I shifting by above 200 mV. This means that, due to the electron withdrawing action of sulfonic groups, the sulfonation makes the reduction easier but the oxidation more difcult, especially the electro-oxidation of the leuco form.

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Fig. 3. In situ thin layer UVvis spectra for solid indirubin (A) and 100 mol dm3 sulfonated indirubin (B) subjected to reduction at E1 followed by re-oxidation at E2 . Buffer pH 1.8.

not considerably higher than under oxygenous condition, suggesting that there would be another faster chemical step leading from the cathodically-generated intermediate with reductive activity to the nal non-electroactive product. Similar spectroelectrochemical measurements were performed for the sulfonated indirubin dissolved in aqueous solutions. The result obtained with pH 1.8 BRS is shown in Fig. 3B, providing a comparison of spectral change with in Fig. 3A. Sulfonated indirubin showed four absorption bands at 249, 290, 361 and 532 nm in the range of 230800 nm (the rst line in Fig. 3B). The former two at smaller wavelengths correspond to the single indole rings as discussed above. The band at 532 nm responsible for the purple-red color corresponds to a typical HOMO LUMO ( *) transition in the conjugated molecules [3]: the HOMO presents a signicant electron density in one of the phenyl rings (the HOMO in indigo lies on the nitrogen atoms and the central C C bond [33]), whereas the LUMO is centered on the single CC bonds and oxygen atoms over the two ve-membered rings as in indigo. Once a reduction potential of 0.20 V was applied (Fig. 3B), the 249 nm band increased in intensity with time accompanied by a blue shift to 236 nm, while the strongest band at 290 nm underwent a decrease in intensity and a blue shift to 286 nm. Both the 361 nm and the 532 nm bands decayed down to near background levels. All these changes show a trend towards the spectrum observed in the reduction of solid indirubin (Fig. 3A), suggesting that the similar leuco product but with the additional sulfonic groups was formed in the reduction of sulfonated indirubin. The 532 nm and 361 nm bands tended to their minimum intensities with time but not to disappearance, indicating the incomplete reduction of the sulfonated indirubin due to the backward chemical oxidation of its leuco product by dissolved oxygen. The subsequent step of potential to 0.54 V led all the four bands to the reverse changes in both wavelength and intensity, indicating the electrochemical and chemical oxidation of the leuco form to the initial keto form. 3.2.2. Buffer pH 7.0 and 9.4 The electro-reduction of solid indirubin in neutral and alkaline buffers led to the products with four adsorption bands around 228, 283, 380 and 580 nm, respectively (Fig. 4). The former two are the same as in the case of pH 1.8 buffer (Fig. 3A), whereas the latter two was newly observed in the neutral and alkaline media. These are two weak and broad bands with larger intensities at pH 9.4 (Fig. 4B) than at pH 7.0 (Fig. 4A). The visible absorption band, which locates in a wavelength range above the maximum absorption wavelength of indirubin (535 nm), suggests a more extensive conjugation in the reduction products than in indirubin. This can be taken as an experimental evidence of leuco-indirubin aggregation in the buffer solutions. There has been a report of indigo aggregation to dimer in chloroform, characterized by the appearance of two new bands around 700 nm and 380 nm [34]. This phenomenon is very analogous to that discussed here. Recently, Fantacci et al. [35] used DFT to simulate hydrogen-bonded indigo dimer and trimer in chloroform, both showing a rather strong intermolecular hydrogen bonding interaction between the carbonyl oxygen and the nitrogen-bound hydrogen. In the present system, the pH of buffer needs to be high enough to ionize the hydroxyl groups of leuco-indirubin for forming the intermolecular hydrogen bonds with the nitrogen-bound hydrogen. Thus, more aggregation of leuco-indirubin was formed at the higher pH of 9.4 (compare Fig. 4B with A and Fig. 3A), along with a rise in the spectral baseline over the whole wavelength range tested (Fig. 4B). This suggests that some dimer/trimer of leuco-indirubin precipitated as solid microparticles suspended in the buffer, scattering the light in all wavelengths. The formation of intermolecular hydrogen bonds led to a decrease in molecular polarity and therefore in the solubility in aqueous solution. Poor solubility of the

3.2. Spectroelectrochemistry of reduction and re-oxidation 3.2.1. Buffer pH 1.8 Spectroelectrochemistry of the indirubin/leuco-indirubin redox system was investigated with the IrCPE in blank BRS. Timedependent UVvis spectra were recorded upon a double potential step triggering the reduction and then re-oxidation of solid indirubin. Fig. 3A shows the spectral curves obtained in pH 1.8 BRS. Reduction of indirubin at 0.32 V resulted in a strong UV absorption band around 230 nm with a weaker one around 283 nm, due to the reduction product(s) striping into the thin-layer BRS from the surface of IrCPE. No absorption was found over the whole visible region in accordance with the leuco product, indicating lack of the conjugation extended over the two indole rings. Indeed, this UV spectrum was similar to that of single indole [32]. After the potential was stepped to 0.36 V triggering the oxidation of leuco-indirubin, the absorption bands mentioned above gradually faded away. The disappearance of these bands indicates that, at the acidic pH 1.8, the formed leuco-indirubin can be oxidized back to the initial insoluble indirubin completely, being inconsistent with the poor electrochemical reversibility reected by the oxidation peak I with a peak current much smaller than its counterpart peak I (Fig. 1A). For this, we conducted an experiment in which the reduction of indirubin was still carried out at 0.32 V but followed by open circuit for the rest of time. The similar spectral fading was observed during open circuit (data not shown), with a rate much slower than that of electro-oxidation at 0.36 V. This means that the cathodically-generated product or intermediate can be chemically oxidized back to indirubin by dissolved oxygen. This chemical oxidation step was seemingly responsible for the low current of re-oxidation peak I compared with the peak I. However, the CVs obtained in anoxic atmosphere gave the peak I

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Fig. 4. In situ thin layer UVvis spectra for solid indirubin subjected to reduction at E1 followed by re-oxidation at E2 . Buffer pH 7.0 (A) and 9.4 (B).

Fig. 5. In situ thin layer UVvis spectra for 100 mol dm3 sulfonated indirubin subjected to reduction at E1 followed by re-oxidation at open circuit. Buffer pH 7.0 (A) and 9.4 (B).

aggregation products was also reected by the low intensities of all the bands compared with observed in acidic buffer. In addition, the precipitated microparticles may block the electrode surface once attached to it. After the potential was subsequently stepped to a given value where oxidation occurred, the absorption bands around 380 and 580 nm became weaker but not disappeared, while those at the smaller wavelengths showed less changes in intensity (Fig. 4B). It is thus clear that the aggregated reduction products could not be oxidized back to the initial solid indirubin (but other lowlyconjugated species), differing from the result observed in the acidic buffer (Fig. 3A). As for the sulfonated indirubin, the spectral changes in pH 7.0 and pH 9.4 buffers are shown in Fig. 5. No new bands were observed during the redox reactions of sulfonated indirubin, showing no aggregation of the reduction products. This is due to the high hydrosolubility of sulfonic group. All the four bands showed their changes in intensity less than observed in the acidic buffer (Fig. 3B), indicating that the sulfonated indirubin was electro-reduced more difcultly in neutral and alkaline media. Accompanied by the backward chemical oxidation of the leuco form by dissolved oxygen, the electro-reduction proceeded up to a low percent conversion to the leuco form, as reected by the intensity decrease rate of the visible band at 532 nm. The kinetic hindrance at the higher pHs may be due to the electrostatic repulsion interaction between the electrode surface and the negatively charged sulfonic groups in indirubin, since the reduction reaction has to be performed at more negative potentials (about 0.7 V vs. Ag/AgCl/KClsat ) in neutral and alkaline media. Some attention should be paid on the band at 361 nm which, upon the reduction reaction, showed a decrease in intensity at pH

1.8 (Fig. 3B), a slight decrease at pH 7.0 (Fig. 5A) but an increase at pH 9.4 (Fig. 5B). In this process the carbonyls on the pyrrole rings were converted to hydroxyls, whose ionization degree and thus the spectrum can be changed by pH adjustment. It can therefore be deduced that the oxygen atoms on the pyrrole rings constituted a part of the chromophore responsible for the band at 361 nm, and the rst pKa for dissociation of the OH protons in sulfonated leuco-indirubin was possibly in the range of 7.09.4, in agreement with the rst pKa ( 8) reported for the leuco-indigo in aqueous environments [7]. After the circuit was opened, the four bands immediately showed reverse changes in both intensity and wavelength (Fig. 5), demonstrating the chemical oxidation of the sulfonated leucoindirubin by dissolved oxygen to the initial keto form. From above, an EC mechanism for the electro-reduction of solid indirubin and sulfonated indirubin is given in Scheme 2. The initial electro-reduction step produces a high reactive carbon free radical through a one-electron one-proton transfer. In the case of solid indirubin, the electron transfer across the electrode/particle interface is coupled with the proton transfer across the electrolyte/particle interface, possibly following the model of an independent and perpendicular diffusion of electrons and cations proposed by Schrder et al. [36]. The resulting carbon free radical then is converted to a keto intermediate by a fast dismutation, and further to the stable enol product (leuco-indirubin) by a keto-enol isomerism. The isomerism of the keto intermediate with reductive activity to the stable leuco-indirubin should be the chemical step responsible for the small anodic-to-cathodic peak current ratio (Fig. 1B). A faster scan rate allows less time for the isomerism of the intermediate to occur, leading to the increase in the anodic-tocathodic peak current ratio with increasing scan rate.

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Fig. 6. In situ thin layer UVvis spectra for solid indirubin (A) and 100 mol dm3 sulfonated indirubin (B) subjected to oxidation at E1 . Buffer pH 1.8.

Scheme 2. An EC mechanism proposed for the reduction and re-oxidation of solid/sulfonated indirubin.

3.3. Spectroelectrochemistry of oxidation 3.3.1. Buffer pH 1.8 Spectroelectrochemistry of the solid indirubin under electrooxidation conditions was investigated with the IrCPE in blank BRS. Fig. 6A shows the time-dependent spectral curves obtained in pH 1.8 BRS. Oxidation of indirubin at 1.0 V resulted in a strong UV absorption band around 239 nm with a very close shoulder at 246 nm as well as a weaker one at 301 nm, being perfectly consistent with the UVvis spectrum of isatin [37]. Accordingly, the indirubin certainly was oxidized to isatin that dissolved into the thin-layer BRS. The central C C double bond of indirubin was broken and could not be reconstituted by reduction at more negative potentials, showing a completely irreversible electro-oxidation process (see Fig. 1B). This behavior is quite different from that of indigo, which can undergo a reversible oxidation reaction producing the dehydroindigo with a central CC single bond, although isatin was also formed at higher oxidation potentials [10].

Fig. 7. In situ thin layer UVvis spectra for solid indirubin subjected to oxidation at E1 . Buffer pH 7.0 (A) and 9.4 (B).

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The spectral changes of the sulfonated indirubin during its electro-oxidation in pH 7.0 and 9.4 buffers (data not shown) are similar to those observed for it in pH 1.8 buffer (Fig. 6B) but at a slower rate, in agreement with the pH effect on the anodic peak current of the sulfonated indirubin (Fig. 1B, the inset). The spectra do not show the rise in baseline as seen in the oxidation of solid indirubin at the same pHs, due to the high hydrosolubility of sulfonic group. From above, an ECE mechanism for the electro-oxidation of solid indirubin and sulfonated indirubin is given in Scheme 3. The two -electrons over the central C C double bond were initially lost generating two carbon cation sites that could be easily attacked by hydroxyl anions/water molecules in the buffer, resulting in the break of the central double bond. Both of the liberated dioxindole radicals would be further oxidized to (sulfonated) isatin through a one-electron and one-proton process. This complex ECE mechanism may lead to different voltammetric features depending mainly on the energy difference between the two electron transfer steps as well as on the rate of the chemical reaction, according to a recent theoretical modeling of surface ECE mechanism due to Gulaboski [41]. The increase in pH level of the buffer has shown a negative impact on the reaction rate (peak current) of electro-oxidation, whether for indirubin or sulfonated indirubin (Fig. 1B). This may be due to the increasing dissociation degree of the NH protons with increasing pH (The pKa of the indole acetate was 5.15 without surfactant [42]). The nitrogen anions might partially compensate the adjacent positive charges generated from the initial electron transfer, stabilizing the carbon cation intermediate and therefore hindering the subsequent reactions. 4. Conclusions The contrastive study of solid indirubin and its sulfonated form gives more comprehensive insight into their electrochemical behaviors and the reaction mechanisms. Similar voltammetric features and main reaction products were observed for the two forms of indirubin but with some reasonable differences. The alkaline pH of electrolyte generally had a negative effect on both the reaction systems, compared with the acidic pH. Electro-reduction of both indirubins produced their leuco forms via an EC mechanism involving dismutation and isomerism reactions of unstable intermediates, resulting in poor electrochemical reversibility. Leuco-indirubins (sulfonated or not) can be oxidized back to the initial reactants by dissolved oxygen. In the alkaline buffers the leuco-indirubin (not sulfonated) may form aggregates with poor solubility in the aqueous solution and poor electrochemical reactivity. Electro-oxidation of both indirubins led to the irreversible formation of isatin (sulfonated or not), probably following an ECE mechanism. The present work shows that the combination of solid state and solution phase spectroelectrochemistry can provide multidimensional information for the reaction mechanism determination. Acknowledgments The authors gratefully acknowledge the nancial support from the National Nature Science Foundation of China (No. 20776033 and 20972038). References
[1] L. Meijer, J. Shearer, K. Bettayeb, Y. Ferandin, Diversity of intracellular mechanisms underlying the anti-tumor properties of indirubins, in: L. Meijer, N.

Scheme 3. An ECE mechanism proposed for the oxidation of solid/sulfonated indirubin.

As for the oxidation of the sulfonated indirubin dissolved in pH 1.8 BRS, the obtained spectra are shown in Fig. 6B. After an oxidation potential of 1.05 V was applied, the 250 nm band underwent an increase in intensity and a blue shift to 240 nm, accompanied by the emergence of a shoulder at 248 nm, whereas the strongest band at 290 nm showed a large decrease in intensity. Both the 361 nm and the 532 nm bands decayed down to near background levels. These changes show a trend towards the spectrum observed in the oxidation of solid indirubin (Fig. 6A), suggesting that the sulfonated isatin was formed in the oxidation of the sulfonated indirubin. The spectra in Fig. 6B very much resemble the UVvis online reaction spectra of sulfonated indigo oxidation by laccase, where the sulfonated isatin was predominantly formed as the nal end product [38]. In addition, superoxide can also convert the sulfonated indigo to the sulfonated isatin [39]. 3.3.2. Buffer pH 7.0 and 9.4 The electro-oxidation of solid indirubin in neutral and alkaline buffers gave rise to the similar absorption bands as in the acidic buffer but with lower band intensities (Fig. 7). The reaction still produced isatin, but only up to a smaller concentration in the buffer with a higher pH. The upward rise in the spectral baseline over the whole wavelength range was observed at pH 7.0 and 9.4, especially at the higher pH, resulting from light scattered by the freshly precipitated particles. Both the low band intensities and the rise in spectral baseline may be due to the poor solubility of the isatin in aqueous alkaline media [40].

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