Investigation of an Electrochemically Switched Heterocyclization

State Key Laboratory of Applied Organic Chemistry (SKLAOC), College of .... as eluent to yield H2Q-SAc as a white solid (0.42 g, 1.55 mmol) in 84% yie...
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Investigation of an Electrochemically Switched Heterocyclization Reaction on Gold Surface Jun Li, Chun-Lin Sun, Lin Tan, Yu-Long Xie, and Hao-Li Zhang* State Key Laboratory of Applied Organic Chemistry (SKLAOC), College of Chemistry and Chemical Engineering, Lanzhou University, 222 Tianshui South Road, Lanzhou, 730000, People’s Republic of China S Supporting Information *

ABSTRACT: We report an investigation of an electrochemically switched heterocyclization reaction on hydroquinone-terminated self-assembled monolayers (SAMs). This reaction involves an electrochemically modulated hydroquinone/benzoquinone transformation step in the SAMs and a subsequent heterocyclization step taking place between the electrochemically generated benzoquinone moieties in SAMs and Lcysteine in solution. The reaction process was monitored by XPS and electrochemical surface-enhanced Raman spectroscopy (EC-SERS). The surface reaction proceeds as a two-step reaction to give a benzothiazine product, which is in contrast to the much more complicated multiple step reactions in solution. This result suggests that the tight molecular packing in the SAMs does not hinder the intramolecular heterocylization reaction, but prevents the intermolecular coupling reaction from happening. This work provides insights to the control and detection of biomolecule related multistep reactions occurring at solid−liquid interface.

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INTRODUCTION Understanding the reactivity of organic molecules immobilized at the solid−liquid interfaces is of fundamental interest and is important for a variety of applications, such as sensing,1,2 solidphase combinational synthesis,3 and heterogeneous catalysis.4 Self-assembled monolayers (SAMs) have been employed in numerous surface reactions since they provide structurally welldefined surfaces at modified electrodes.5−7 Because the structure of electrode−liquid interface plays an important role in surface chemistry and biological systems, investigations on the interfacial reactions taking place on SAMs have attracted a great deal of attention from both fundamental and practical perspectives.8−10 Quinone derivatives participate in many biological processes and are also useful building blocks for organic materials.11,12 Quinone-terminated self-assembled monolayers (SAMs) on electrode surfaces have been highlighted for chemical reactions at interfaces. Benzoquinone undergoes a reversible two-electron reduction to give the corresponding hydroquinone and therefore permits use of cyclic voltammetry to measure the loss or change of benzoquinone moiety on the surface. Recently, chemical reactions between benzoquinone and other biomolecules have been studied for immobilizing biomolecules onto solid supports, which is important for a wide variety of applications ranging from biochips to model substrates for biological studies.13,14 Several surface reactions involving surface immobilized benzoquinones have been studied electrochemically, including interfacial Diels−Alder reactions,15 amine or thiol additions,8,9,16 and enzymatic reactions.10 © XXXX American Chemical Society

Among the various surface characterization techniques used to study the surface chemical reactions,15,17−19 Surface Enhanced Raman Scattering (SERS) is particularly competent for the chemical reactions on noble metal surface. Over the past three decades, SERS has developed into a powerful surface diagnose technique for investigation on electrode surface, which can be readily combined with other techniques to conduct in situ spectroscopic characterization and real-time monitoring.19−22 In particular, the EC-SERS technique has been used in previous investigations on the surface chemistry of various organic functional groups, including quinone derivatives. For example, Wu et al. investigated the adsorption and reduction reactions of an anthraquinone derivative on gold electrodes. Long et al. used EC-SERS to monitor the interconversion between NAD+ and NADH mediated by ubiquinone in a lipid bilayer membrane. Previous works on the surface reactions of quinone SAMs have focused on the recognition,11 kinetics, and structure effects8,23−25 of a few simple one step reactions. However, investigations on more complicated multistep surface reactions are very rare. Wolfram et al.26 have reported that benzoquinone could react with L-cysteine in aqueous solution, in which the Lcysteine first reacts with benzoquinone through Michael addition, and the resulting adduct then undergoes an intramolecular cyclization to give heterocycles containing 1,4benzothiazine structure. Meanwhile, 1,4-benzothiazine derivaReceived: January 24, 2013 Revised: April 1, 2013

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dropwise via a cannula to a second flask containing a dry THF (26 mL) solution of 1,6-dibromododecane (1.3 g, 54.3 mmol) at 0 °C. The contents were stirred for 30 min at 0 °C, and the reaction was allowed to warm up to room temperature. The mixture was stirred overnight at room temperature, diluted with 130 mL dichloromethane, poured into 65 mL saturated NH4Cl solution and washed with brine (80 mL). The solution was extracted with dichloromethane (3 × 20 mL), and the organic solution was washed with water and dried over MgSO4. The solvent and unreacted dimethoxybenzene were evaporated in vacuum, and the residue was purified by column chromatography using silica gel and 90:10 hexane:ethylacetate as eluent to yield 1 as colorless oil (2.82 g, 9.41 mmol) in 52% yield. 1H NMR (CDCl3): dH 1.36−1.46 (br, 4H, aliphatic CH2), 1.58 (quintet, 2H, −CH2CH2CH2Br), 1.85 (quintet, 2H, −CH2CH2Ph), 2.58 (t, J = 7.8 Hz, 2H, −CH 2Ph), 3.39 (t, J = 6.3 Hz, 2H, −CH2Br), 3.76 (s, 3H, −OCH3), 3.77 (s, 3H, −OCH3), 6.66−6.77 (m, 2H, Ar), 6.83 (s, 1H, Ar). 13C NMR (CDCl3) 27.97, 28.53, 29.56, 30.08, 32.72, 33.98, 55.55 (−OCH3), 55.83 (−OCH3), 110.50, 111.05, 116.19, 132.19, 151.66, 153.32. S-6-(2,5-Dimethoxyphenyl)hexyl Ethanethioate (2). To a 100 mL round-bottom flask fitted with a condenser were added 1 (0.78 g, 2.6 mmol), potassium thioacetate (0.59 g, 5.2 mmol), and 30 mL of acetone. The reaction mixture was heated to reflux and stirred for 6 h. The completion of reaction was monitored by TLC. The solvent was then removed in vacuum, and water (10 mL) and toluene (20 mL) were added. After separation, the organic phase was washed with water (2 × 10 mL), dried over MgSO4, transferred to a round-bottom flask via gravity filtration and concentrated to yield 0.55 g of 2 (yield = 71%). 1H NMR (CDCl3): dH 1.36−1.40 (br, 4H, aliphatic CH2), 1.55−1.59 (m, 4H, aliphatic CH2), 2.32 (s, 3H, −CH3), 2.57 (t, J = 8.1 Hz, 2H, −CH 2Ph), 2.86 (t, J = 7.8 Hz, 2H, −CH2−S), 3.76 (s, 3H, −OCH3), 3.77 (s, 3H, −OCH3), 6.69−6.78 (m, 2H, Ar), 6.84 (s, 1H, Ar). 13C NMR (CDCl3) 28.66, 28.95, 29.14, 29.42, 29.64, 30.14, 30.62, 55.70 (−OCH3), 55.89 (−OCH3), 110.52, 111.09, 116.19, 132.36, 151.70, 153.35, 196.11. S-6-(2,5-Dihydroxyphenyl)hexylethanethioate (H2Q-SAc). Compound 2 (0.55 g, 1.84 mmol) was dissolved in dry methylene chloride (30 mL) in a round flask immersed in the ice bath under nitrogen and temperature cooled down to −75 °C with dry ice/acetone mixture. A solution of BBr3 (1.0 M in dry dichloromethane, 10 mL, 10.4 mmol) was added dropwise to the solution of 2. The mixture was allowed to warm up to room temperature and stirred for an additional 2 h. The solution was poured into 25 mL of saturated NH4Cl solution and was extracted with CH2Cl2 (3 × 20 mL), and the organic phase was washed with water and brine and then dried over MgSO4. The solvent was removed in vacuum, and the residue was purified by column chromatography using silica gel and hexane:diethyl ether (70:30) as eluent to yield H2Q-SAc as a white solid (0.42 g, 1.55 mmol) in 84% yield. 1H NMR (CDCl3): dH 1.34−1.37 (br, 4H, aliphatic CH2), 1.54−1.60 (m, 4H, aliphatic CH2), 2.33 (s, 3H, −CH3), 2.54 (t, J = 7.5 Hz, 2H, −CH 2Ph), 2.86 (t, J = 7.8 Hz, 2H, −CH2−S), 4.63 (br, 2H, −OH), 6.66−6.77 (m, 2H, Ar), 6.83 (s, 1H, Ar). 13C NMR (CDCl3) 28.47, 28.61, 29.10, 29.21, 29.32, 29.76, 30.68, 113.27, 116.04, 116.77, 129.80, 147.29, 149.33, 197.42. Preparation of Gold Electrode and SERS-Active Substrate. The gold electrode surface was first mechanically polished with alumina powders of 0.3, 0.1, and 0.05 μm, consecutively, until a mirror-like surface was obtained, followed by sonication in water. Then the electrode was rinsed with ultrapure water, and sonicated to remove any adhering alumina. After that, the gold electrode was cleaned for 10 min in piranha solution (H2O2 + H2SO4, 3:7 v/v). Subsequently, the gold electrode was electrochemically cleaned in 0.5 mol L−1 H2SO4 solution in the potential range of −0.2 and 1.5 V vs SCE (the same with the following electrochemical experiments). After rinsing with ultrapure water, the gold electrode was kept at −0.3 V in 0.1 mol L−1 KCl until the stabilization of the current. Then, the potential was scanned to 1.2 at 1 V s−1, set for 1.2 s for oxidation, then scanned back to −0.3 at 0.5 V s−1 and set for 30 s for reduction. The cycle was repeated for 25 times and the final potential should be −0.3 V to ensure a reduced state of the electrode. This roughening process resulted in an Au surface with a brown appearance.21 For the XPS and

tives are known to exert many bioeffects in vivo and in vitro;27 therefore, they are under investigation for their potential applications in the fight against neurodegenerative diseases28,29 and cancer.30,31 A 1,4-benzothiazine terminated surface may find applications in fields like biosensing and drug screening assays. However, the 1,4-benzothiazine produced by reaction between benzoquinone and L-cysteine in solution phase could undergo a series of intermolecular coupling reactions, and give several complicated products. Therefore, a method that is able to control the progress of the reaction is of practical importance. Herein, we investigate the chemical reaction between Lcysteine and the benzoquinone moieties immobilized on gold surface with the assistance of EC-SERS technique (Scheme 1). Scheme 1. Illustration of the Reaction Taking Place between the Benzoquinone Immobilized on Gold Surface and LCysteine

To the best of our knowledge, literatures dealing with such multistep reaction taking place at liquid/solid interface are very rare. The motivation is to understand two fundamental issues. First, is it possible to utilize the electrochemical reactivity of benzoquinone unit to control the progress of an interface reaction? Second, is the multistep reaction take place on SAMs different from that in solution, since the spatial hindrance from the tightly packed monolayer structure may restrict the intramolecular cyclization and the intermolecular coupling steps?

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EXPERIMENTAL SECTION

Materials. All of the reagents and materials were purchased from J&K Chemical Co. (China), and are of analytical grade. The solvents were purified by the standard procedures. All aqueous solutions were prepared with ultrapure water obtained with a Milli-Q System (Billerica, MA, U.S.). All of the experiments were carried out at room temperature, unless stated otherwise. Synthesis. The S-6-(2,5-dihydroxyphenyl)hexylethanethioate (H2Q-SAc) was synthesized following the procedure reported by Hickman et al. with modification.32 2-(6-Bromohexyl)-1,4-dimethoxybenzene (1). A solution of nbutyllithium (2.5 M in hexane, 20.2 mmol) was slowly added to a dry THF (65 mL) solution of 1,4-dimethoxybenzene (2.5 g, 18.1 mmol) in a nitrogen purged Schlenk flask over 20 min. The mixture was stirred for an additional 60 min at room temperature and transferred B

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ellipsometry measurements, 25 × 75 mm2 gold coated glass slides were used as the substrates. The glass slides were coated by thermal evaporation with a 50 Å chromium adhesion layer followed by a 500 Å gold layer (99.99%). Preparation of SAMs. The gold substrate was modified with SAM of HQ-SH (deprotection product of HQ-SAc) by immersion in 10−4 M solution of H2Q-SAc, in slightly acidified EtOH, for 24 h at room temperature. The modified electrodes were further rinsed with ethanol to remove physically adsorbed molecules prior of further characterization. Electrochemical Experiments. The electrochemical behaviors of the H2Q-SH SAMs on gold electrode were studied in 0.2 M phosphate buffer solution (PBS) of pH: 6.0 using a CHI660 (CHI, U.S.) electrochemistry workstation. The EC-SERS experiments were performed in an EC-SERS cell (as shown in the Scheme 2), in the

monolayers was assumed to be isotropic with a refractive index n equal to 1.5. Computational Details. The geometry optimization and Raman spectra simulation of the different structures were calculated by using the Density Functional Theory (DFT) with B3LYP method, using the basis set of 6-311++G(d,p). All quantum chemistry calculations were performed by Guassian09.33 EC-SERS Experiments. The EC-SERS experiment setup is shown in the Scheme 2.21 SERS spectra were obtained using a confocal microscope Raman system (Renishaw in via Raman microscope) equipped with a Renishaw CCD camera (Scheme 2). The microscope is based on a Leica DM2500 M system and uses a 50× long-workinglength objective so that the objective will not be in contact with the electrolyte. A holographic notch filter was used to filter the exciting line and two selective holographic gratings (1200 g/mm, 2400 g/mm) were employed depending on the spectral resolution required. The exciting wavelength was 785 nm.

Scheme 2. EC-SERS Experiment Setupa

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RESULTS AND DISCUSSIONS In this work, the compound 2-(6-mercaphexyl)benzene-1,4-diol (H2Q-SH), was used as an electroactive probe, which was covalently attached onto the gold surface. Figure 1 shows the

a

Figure 1. Voltammograms of a H2Q-SH monolayer in PBS (pH: 6.0) for different scan rates (from 0.1 to 0.9 V s−1). The inset shows the linear dependence of the current on the potential scan rate to highlight the presence of a surface confined redox species.

WE: gold electrode; CE: platinum wire; RE: silver wire.

three electrodes geometry, using a gold working electrode, a Pt counter electrode, and a silver wire as reference electrode. While other voltammetric experiments were carried out with an SCE reference. For impedance studies, a sine wave with 5 mV of amplitude was applied to the electrode over the formal potential of the redox couple (0.2 V). Impedance spectra were collected in the frequency range between 105 and 10−1 Hz. All of the electrochemical experiments were carried out at room temperature under ambient conditions. Each electrochemical spectrum was initiated at the negative potential limit following a 2 s quiescence period. X-ray Photoelectron Spectroscopy (XPS). XPS spectra were acquired on a Perkin-Elmer Phi-5500 spectrometer operating at a base pressure of ∼10−9 Torr and equipped with an Al KR source (E = 1486.6 eV) and quartz monochromator. All spectra were acquired with a resolution of 0.125 eV except surveys, which had a resolution of 0.4 eV. The Shirley function was used as a background and Gaussian− Lorentzian cross-products used to fit the individual peaks. The samples for XPS measurements were prepared from the SAMs before and after being reacted with L-cysteine on gold-coated glass chips (5 ×5 mm size). Before the chips were incubated in solutions of the SAMs, the chips were carefully precleaned by sonication in Millipore H2O three times. Ellipsometry. A commercially available imaging ellipsometry (EP3, Nanofilm Technologie, Germany) was used to determine the thicknesses of the monolayers on gold substrate. The measurements were taken using a 532 nm 20 mW solid state laser source. The light incident angle was 70° to the surface normal. The organic phase of the

cyclic voltammograms (CVs) redox waves associated with the 2e−-2H+ redox reaction of the benzoquinone moieties in the H2Q-SH monolayer, in 0.2 M PBS (pH: 6.0) at different scan rates from 0.1 to 0.9 V s−1. The redox peak current scales linearly with the voltage scan rate (inset to Figure 1) as expected for a surface bound redox species. The surface coverage (Γ, mol cm−2) of H2Q-SH on the gold surface was obtained from eq 1: Γ = Q /nFA

(1)

where Q is the charge obtained by integrating the anodic or cathodic peaks in CVs; n is the electron transfer number, which is 2; F is the Faraday constant; and A is the surface area of the gold electrode. The calculated electrochemical surface area indicated a roughness factor of the 1.59 of the gold electrode. The coverage for the SAM of H2Q-SH is then calculated to be 2.82 × 10−10 mol cm−2, which is consistent to the typical coverage found for the hydroquinone/quinone redox couple with bridges containing alkane thiols reported in the literature.34 Figure 2(a) shows a set of consecutive CVs for H2Q-SH monolayer in 0.2 M PBS (pH: 6.0) after addition of L-cysteine solution. In the absence of L-cysteine, the hydroquinone groups underwent an oxidation process at +30 mV (versus SCE) and C

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Figure 2. (a) Sequence of CVs of a H2Q-SH monolayer after the addition of 2 mL 10−3 M L-cysteine solution in 10 mL PBS (pH: 6.0) (b) CVs of SAM of H2Q-SH recorded in 0.2 M PBS (pH: 6.0) after addition of 2 mL 10−3 M L-cysteine solution in 10 mL 0.2 M PBS (pH: 6.0) for 5 min under different potentials: OCP (open circuit potential), −0.2 V and +0.2 V, respectively.

the resultant benzoquinone groups can be reduced at −24 mV, and the voltammetric response is reversible. The consecutive CVs in the solution of L-cysteine show that both the anodic and cathodic peaks decreased with time and also the peak-to-peak separation increased due to kinetic limitations. Given the fact that L-cysteine contains thiol group and may also form SAMs on gold surface, a series of control experiments were conducted to check the possibilities of the H2Q-SH molecules being desorbed from the gold surface or replaced by the L-cysteine on the above electrochemical condition (Figure S2 of the SI). It is found that the SERS spectra of an inert SAM of n-octadecyl thiol do not change after the addition of 10−3 M L-cysteine solution. It is then concluded that, under the given electrochemical conditions, the monolayer structure cannot be changed by the addition of L-cysteine. Besides, the Figure S3 of the SI demonstrates that the SERS spectrum of L-cysteine monolayer on gold electrode is different from the spectrum of the monolayer after the surface reaction, which indicates that the replacement of H2Q-SH by L-cysteine does not happen. Therefore, the gradual decline in the peak currents presented in Figure 2(a) can only be ascribed to the chemical reaction taking place between the H2Q-SH monolayer and the L-cysteine in solution. Previous study on the Michael addition between benzoquinone and thiol moieties showed shift of the redox waves.8 In this work, the reaction between H2Q-SH monolayer and L-cysteine results in the total disappearance of the redox waves, suggesting that the reaction proceeded to the intramolecular heterocyclization step to give the electroinactive 1,4benzothiazine product, as shown in Scheme 1. It is noted that such peak disappearance phenomena makes it easier to monitor the surface reaction than the previously reported peak shift situation. Figure 2b shows the CVs recorded in 10 mL 0.2 M PBS (pH: 6.0) 5 min after the addition of 2 mL 10−3 M L-cysteine solution while the electrode was held at −0.2 V. After rinsing the electrode with ultrapure water, a CV was recorded in 0.2 M PBS (pH: 6.0), which was almost the same as the initial CV (Figure 2b). This result argues that the headgroups of the monolayer presents as hydroquinone at −0.2 V, which do not react with L-cysteine. In contrast, after being held at +0.2 V in PBS for 5 min, addition of L-cysteine solution resulted in complete disappearance of the redox peaks. Figure 2 confirms that the surface immobilized hydroquinone moieties can be electrochemically converted to benzoquinone and then react with L-cysteine in solution to give electroinactive benzothiazine structure. Electrochemical impendence spectroscopy (EIS) was employed to assess the structural integrity of the monolayer.35 The

resulting Nyquist diagrams for the H2Q-SH modified gold electrode before and after the electrochemically enabled reaction with L-cysteine is provided in Figure 3. The

Figure 3. Nyquist plots for the H2Q-SH monolayer on gold electrode before and after the reaction of the benzoquinone with the L-cysteine in 0.2 M PBS (pH: 6.0) at 0.2 V vs SCE, in the presence of 1 mM Fe(CN)63−/4− probe. The lower inset is the Nyquist plot of bare gold electrode on the same condition. The upper inset is the schematic representation of equivalent circuit used to obtain electrochemical parameters of SAMs in presence of 1 mM Fe(CN)63−/4− probe.

impendence plots are characterized by two distinct regions: (I) A semicircle in higher frequency related to charge transfer process, which was electrically described by a resistance in parallel with a capacitor related to the charge-transfer and electrode/SAM double layer, respectively. (II) A 45° line in the complex-plane impendence plot defined a Warburg region of semi-infinite diffusion of species in the modified electrode. For a bare gold electrode, a very small semicircle (1−400 Hz) followed by a straight Warburg line was observed, indicating a diffusion controlled process. In contrast, the SAMs of H2Q-SH showed a clear semicircle. After the reaction with L-cysteine, the diameter of the semicircle dramatically increased, implying a higher barrier for electron transport. More quantitative information about the monolayer structures was obtained by fitting the impendence data to the circuit presented in the inset of Figure 3. The resistance (2.14 KΩ) of the monolayer after the reaction was found to be about twice of the HQ-SH SAMs (1.02 KΩ). This increase in resistance value is ascribed to less solvent penetration in the monolayer after the reaction. The difference in the dielectric D

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spectrum of the HQ-SH SAMs presents two components located at 161.7 and 162.9 eV with an area ratio of 2:1 and a separation of 1.2 eV, which can be assigned to the S 2p3/2 and S 2p1/2 peaks of the sulfur element.36 After reacting with Lcysteine, the SAMs show an increased sulfur signal, which is approximately twice that as before the reaction, consistent with the formation of the benzothiazine units. An O peak is observed at 532.2 eV in the spectrum acquired before the reaction. In contrast, two O species are distinguished after the reaction. The peak at 533.4 eV is assigned to the carboxyl O, while the 531.7 eV peak is attributed to the more electron rich carbonyl group. The area ratio of these two peaks is 1:2, which is consistent with that expected for the product shown in Scheme 1. As proposed above, the headgroups of the H2Q-SH monolayer present as hydroquinone under negative potentials and as benzoquinone under more positive potentials. To verify this hypothesis, EC-SERS experiments were conducted to monitor the changes of the head groups under different electrochemical conditions. Figure 5 shows the SERS spectra

properties can also be inferred from the capacitance values of each SAMs. The capacitance of the HQ-SH SAMs was 1.56 μF cm−2, whereas that of the SAMs after the reaction was 1.20 μF cm−2. The capacitance of a monolayer is given by C = εrε0A/d, in which C is the capacitance, εr is the dielectric constant of the SAMs, ε0 is the permittivity of free space, A is the surface area, and d is the thickness of the monolayer. Therefore, for the systems presented here there are two factors that are important with regard to the magnitude of the measured capacitance: the dielectric constant of the SAMs, which is directly proportional to the capacitance; and the thickness of the film, which is inversely proportional to the capacitance. We attribute the lower capacitance of the HQ-SH SAMs after reacting with Lcysteine to the increase in film thickness. To verify whether the surface reaction changes the monolayer thickness, ellipsometric measurements were performed on the SAMs. It is found that the thickness of the H2Q-SH monolayers on gold is about 16 ± 1.5 Å. After reacting with L-cysteine, the film thickness increase to 20 ± 1.5 Å. Such thickness data are consistent with the expected molecular lengths changes before and after the surface reaction (Scheme 1). X-ray photoelectron spectroscopy (XPS) analysis was conducted on the HQ-SH SAMs before and after the reaction (Figure 4), which provided important information regarding

Figure 5. SERS spectra of a H2Q-SH monolayer on gold electrode while applying different potentials: +0.6 V and −0.4 V.

acquired under two different potentials in 0.2 M PBS (pH: 6.0) on a SERS-active gold electrode. The potential was scanned from −0.4 V to +0.6 V, such that hydroquinone evolved from its reduced state (hydroquinone) to fully oxidized state (benzoquinone). The benzene ring structures of hydroquinone and the carbonyl group (CO) of benzoquinone have unique spectroscopic signatures6,37 and yield Raman spectral changes in both intensity and peak position. The spectrum acquired at −0.4 V (Figure 5a) shows an intense SERS band at 1613 cm−1. The peak position is consistent with the aromatic CC stretching band for H2Q-SAc powder (Figure S4 of the SI) at 1622 cm−1 and the anthraquinone derivative reported in literature.6 The SERS bands change significantly when the potential is changed to +0.6 V, which shows a clear peak due to the carbonyl group at the position of 1667 cm−1, indicating that headgroups of the monolayer have transformed from hydroquinone to benzoquinone. After applying a reducing potential of −0.4 V, the SERS spectrum returns to that corresponding to hydroquinone, indicating that electrochemically controlled transformation between benzoquinone and hydroquinone species was highly reversible. The reaction between the electrochemically generated benzoquinone moieties and the L-cysteine in solution is then monitored by EC-SERS. At the beginning, the SAM was first

Figure 4. XPS spectra of the SAMs before and after reacting with Lcysteine.

the chemical transformation of the SAMs. The C 1s signal of the HQ-SH SAMs gives a principal peak at 284.8 eV, characteristic of the alkyl part of the SAMs. After reacting with L-cysteine, a new component centered at 287.7 eV is detected, this can be assigned to carbonyl moiety from the product. No apparent peak is observed in the N 1s region before the reaction, consistent with the fact that the HQ-SH molecule contains no nitrogen atoms. In contrast, after the reaction with L-cysteine, a weak but detectable peak appears at 400 eV, implying the presence of N in the product. The S 2p E

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chemical conditions were monitored by EC-SERS. Figure 7 shows the SERS spectra obtained on different gold electrodes

held at +0.6 V so that the headgroups were all converted to benzoquinone. Then a solution containing 10−3 M L-cysteine was added. Sequence of time-resolved SERS spectra were acquired during the reaction as presented in Figure 6. Figure

Figure 7. SERS spectra of gold electrodes that were held at different potentials for 4000s in the solution containing 1.6 × 10−4 M L-cysteine (PBS, pH: 6.0).

Figure 6. Sequence of SERS spectra on a SERS-active gold electrode during the reaction. The reaction took place on the H2Q-SH modified gold electrode after the addition of 10−3 M L-cysteine under +0.6 V. (a) The gradual decrease of the peak intensity at the position of 1667 cm−1 (b) The gradual increase of the peak intensity at the positions of 1011 cm−1,1131 cm−1.

that were held at various potentials for 4000 s in the solution containing 1.6 × 10−4 M L-cysteine (PBS, pH: 6.0). We can see that under potentials between −0.5 and 0 V, the SERS spectra did not change very much, and the spectra correspond well to the hydroquinone state, as shown in the Figure 5. However, the peak at 1011 cm−1 started to increase with the potential move toward more positive values than 0.1 V. A further increase in the positive potential beyond 0.5 V did not increase the peak intensity any more, indicating the completion of the reaction. These results confirm that under potentials between −0.5 and 0 V, the monolayer exists as hydroquinone, which does not react with L-cysteine. From 0.1 V, the headgroups of the monolayer begin to be transformed into their oxidized state, which can react with the L-cysteine.

6(a) illustrates that there are three new peaks rising gradually with the reaction time, indicating new compounds are generated on the SAM surface. The new peak at 1011 cm−1 may be assigned to the C−O stretching mode and the peak at 1131 cm−1 is assigned to the vibration of CN stretching. To assist the assignment of the Raman spectra, we have simulated the Raman spectrum of the expected product. Comparison between the observed and simulated spectra confirms that the new peaks are due to the production of benzothiazine on the surface (Figure S5 of the SI). The simulated spectrum also indicates that the carboxyl group of the benzothiazine product will occur at around 1719 cm−1, but it can barely be observed experimentally due to the very small Raman cross section. Figure 6b more evidently shows that the reaction taking place on the carbonyl site of the quinone species. It can be seen that the peak intensity at 1667 cm−1, which corresponds to the quinone CO stretching, decreased gradually with the reaction time. Importantly, the peak at 1667 cm−1 did not vanish completely even with prolonged reaction time, indicating that there were still carbonyl groups remaining in the product. This result is consistent with the fact that the benzothiazine product has only one carbonyl group, which is one carbonyl group less than that of benzoquinone. The reason that the remaining CO intensity is less than half of the initial intensity may be attributed to the orientational change of the CO groups after the reaction. As mentioned above, the reaction between L-cysteine and benzoquinone could further proceed to yield complicated products in solution which do not possess any carbonyl moieties.26 Therefore, it can be concluded that reaction between the L-cysteine and benzoquinone in the SAMs stops at the benzothiazine step and does not proceed further to intermolecular coupling products. This result is in good agreement with the XPS results. As discussed above, the headgroups of the monolayer undergo interconversion between benzoquinone and hydroquinone states with the changes of electrode potentials. The transformations of the headgroups under different electro-

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CONCLUSIONS We have successfully realized the occurrence of a relatively complicated multistep reaction between the electrochemically generated benzoquinone moieties on gold electrode and Lcysteine in solution. The occurrence of the benzothiazine product on the surface has been confirmed by CV, EIS, XPS, and EC-SERS measurements. By utilizing the electrochemical controlled reactivity of the benzoquinone immobilized on the electrode surface, the interfacial reaction can be electrochemically switched, with the reaction progress well monitored. Importantly, the interfacial reaction differs markedly from the reaction of the same reactants in solution due to the spatial restriction in the densely packed monolayers. It is believed that this work may provide a broad prospect for the control and detection of biologically relevant multistep surface reactions. Further research may include exploration of the applications of the surface bound benzothiazine moieties in applications like surface recognition or biosensing.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and Raman spectra of different samples. This material is available free of charge via the Internet at http://pubs.acs.org. F

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: (+86) 931 8912365; e-mail: [email protected]. cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Basic Research Program of China (973 Program) No. 2012CB933102, National Natural Science Foundation of China (NSFC. 21233001, 21190034, 21073079, J1103307), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP. 20110211130001), the Fundamental Research Funds for the Central Universities and 111 Project.

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