Synthesis and Characterization of Ferrocene Derivatives and Preliminarily Electrocatalytic Oxidation of L-Cysteine at Nafion-Ferrocene Derivatives Modified Glassy Carbon Electrode

Five new structural ferrocene derivatives (2a∼2e) were firstly synthesized and characterized by H NMR, C NMR, ESI-MS, and XRD. Subsequently, the preliminarily electrocatalytic oxidation of L-cysteine (L-Cys) at nafion-ferrocene derivatives modified glassy carbon electrode (GCE) has also been investigated by cyclic voltammetry. The results showed that 2e can dramatically electrocatalyze the oxidation of L-cysteine at itsmodifiedGCE in 0.1mol LNaNO 3 aqueous solutionwith a quasireversible process with ΔE p ≈ 55mV.

L-Cysteine (L-Cys) plays a crucial role in both bioand environmental chemistry and can be applied in many biochemical processes and diagnosis of disease states.Especially, L-Cys provides a modality for the intramolecular crosslinking of proteins through disulfide bonds to support their secondary structures and functions.Therefore, it is very important to develop simple and effective methods to trace L-Cys detection.Many methods, such as spectrometric method [17], chromatography [18], and electrochemical method [19][20][21][22][23], have been used to detect the trace content of L-Cys.
However, electrochemical method has attracted considerable attention for simple operation, fast response, and sensitive in situ detection.
The oxidation of L-Cys at Hg, Au, Ag, Pt, and diamond electrodes has been reported [24][25][26][27].However, the direct oxidation of L-Cys at GCE is very sluggish.Ferrocene derivatives have been used as the selective probes for detecting the trace ions, biomacromolecules [28][29][30][31][32]. Nafion is a special material, which possesses widespread applications in the field of electrochemistry analysis, chemical sensor, and nanomaterials [33,34].According to our survey, no electrocatalytic oxidation of L-Cys at the nafionferrocene derivatives modified GCE has been reported.In this work, five new structural ferrocene derivatives (2a∼ 2e) (Scheme 1) were firstly synthesized and then confirmed by IR, 1 H NMR, 13 C NMR, MS, and XRD.Subsequently, their preliminarily electrocatalytic oxidation of L-Cys at the nafion-ferrocene derivatives modified GCE has also been investigated.Scheme 1: Synthetic routes of ferrocene derivatives.[35].Ferrocene carboxylic acid 1 (0.46 g, 2 mmol) and DCC (0.453 g, 2.2 mmol) were added into a 100 mL one necked round bottom flask with 20 mL dry THF.This mixture was stirred under cold bath and a THF solution (10 mL) containing DMAP (0.268 g, 2.2 mmol) was added dropwise by syringe, the mixture was stirred under cold bath for 30 min, and then temperature rose naturally to room temperature.The completion of reaction was judged from the simple TLC analysis.The mixture was evaporated under reduced pressure and the residual directly purified by column chromatography (EtOAc/petroleum ether: 5 : 1 to

Synthesis of Ferrocene Carboxylic Acid Benzotriazol-1yl Ester (2b).
Ferrocene carboxylic acid 1 (0.46 g, 2 mmol), DCC (0.453 g, 2.2 mmol), and HOBt (0.337 g, 2.5 mmol) were added into a 100 mL one necked round bottom flask with 20 mL dry THF.This mixture was stirred under cold bath and a THF solution (10 mL) containing DMAP (0.268 g, 2.2 mmol) was added dropwise by syringe, the mixture was stirred under cold bath for 30 min, and then temperature rose naturally to room temperature.The completion of reaction was judged from the simple TLC analysis.The mixture was evaporated under reduced pressure and the residual purified directly by column chromatography (EtOAc/petroleum ether: 5 : 1 to 2 : 1) to give the desired compound 2b.

Synthesis of Ferrocene Carboxylic Acid 4,6-Dimethoxy-[1,3,5]triazin-2-yl Ester (2e).
Ferrocene carboxylic acid 1 (0.46 g, 2 mmol) and CDMT (0.438 g, 2.5 mmol) were added into a 100 mL one necked round bottom flask with 20 mL dry THF.This mixture was stirred under cold bath and a THF solution (10 mL) containing NMM (0.253 g, 2.5 mmol) was added dropwise by syringe, the mixture was stirred under cold bath for 30 min, and then temperature rose naturally to room temperature.The completion of reaction was judged from the simple TLC analysis.The mixture was evaporated under reduced pressure and the residual purified directly by column chromatography (EtOAc/etroleum ether: 5 : 1 to 2 : 1) to give the desired compound 2e.
Light yellow solid (0.863 g, 92% yield  and 50 L 5% nafion solution were added in a 2 mL centrifugal tube, then 1 mL of absolute alcohol was added, and the mixture was sonicated for 10 min to obtain the solution of 1.5 × 10 −2 mol L −1 2a.
The detailed process for preparing the solutions of 1.5 ×10 −2 mol L −1 2b, 2c, 2d, and 2e is the same as 2a.

General Process for Preparing the Modified Glassy Carbon Electrode and Preliminarily Electrocatalytic Oxidation of L-Cysteine.
A GCE was polished with 0.1 m -Al 2 O 3 , rinsed with doubly distilled water, sonicated in 1 mol L −1 H 2 SO 4 for 5 min, and rinsed with doubly distilled water again prior to each experiment.Then, it was dried naturally.
A modified GCE was prepared by placing 5 L of solution prepared in Section 2.3.1 onto the dried GCE surface by microsyringe.The electrode was dried naturally to obtain the modified GCE.
The modified GCE, a twisted platinum wire electrode, and Ag/AgCl electrode were dipped into 20 mL (V/V, 1 : 1) water solution of 6.0 × 10 −3 mol L −1 L-Cys and 0.1 mol L −1 NaNO 3 .The Ag/AgCl electrode was connected to the main body of the cell through a luggin capillary whose end was connected on the modified GCE and positioned close to the electrode surface.The solution was thoroughly flushed with high purity nitrogen for 5 min before each run to remove the oxygen from the solution in the electrochemical cell.All experiments were carried out at room temperature.

Results and Discussion
3.1.Chemistry.Ferrocene derivatives (2a∼2e) were firstly synthesized in higher yield and their structures were confirmed by IR, 1 H and 13 C NMR, and mass spectrometry.
There showed three kinds of proton signals for ferrocene core of the ferrocene derivatives (2a∼2e) in 1 H NMR spectra (Figure 2), the protons of C 5 H 5 showed a singlet, the C 5 H 4 showed two triplets of 2a∼2c, but the C 5 H 4 showed two

X-Ray Crystallographic
Study.X-ray diffraction data for 2c was collected on a Siemens Smart CCD diffractometer equipped with a graphite-monochromated CuKa radiation ( = 1.54180 >).The structure was solved by direct method using the program SHELXL-97 and refined by full-matrix leastsquares techniques on  2 with SHELXL-97 program package [36,37].The molecular structure (Figure 3) and crystal data of 2c are listed in Tables 1 and 2.

Cyclic Voltammetric Behavior of Compounds 2a∼2e and Nafion Modified GCE Electrocatalyze the Oxidation of L-Cys.
The preliminarily electrochemical oxidation of L-Cys with the nafion-ferrocene derivatives modified GCE in 0.1 mol L −1 different supporting electrolyte solutions, such as NaCl, NaNO 3 , NaAc, and Na 2 SO 4 , has been investigated by cyclic voltammetry.The results (Figure 4) showed that 2e can promote the electrochemical oxidation of L-Cys dramatically in 0.1 mol L −1 NaNO 3 solution with a quasireversible process with Δ  ≈ 55mV (curve (d) in Figure 4), while direct oxidation of L-Cys at a GCE is very sluggish (cruve (b) in Figure 4).

Conclusion
In this study, five ferrocene derivatives (2a∼2e) were firstly synthesized and structurally characterized using spectroscopic methods.The preliminarily electrochemical oxidation of L-Cys with the nafion-ferrocene derivatives modified GCE in 0.1 mol L −1 NaNO 3 solution has also been investigated by cyclic voltammetry.The results showed that 2e can promote the electrochemical oxidation of L-Cys dramatically at its modified GCE with a quasireversible process with Δ  ≈ 55 mV, while L-Cys itself showed a sluggish electrochemical response at GCE.Thus, 2e modified GCE can be used to detect the trace L-Cys content.(The quantitative analysis of trace L-Cys and electrochemical kinetics of 2e modified GCE are undergoing.)Based on this research, more ferrocene derivatives will be synthesized and the oxidation of L-Cys and other biological molecules at the modified GCE will be studied soon.

Table 1 :
Crystallographic data and structure refinement for 2c.Three signals of the carbon atoms of C 5 H 4 and one signal of carbon atom of C 5 H 5 appear in 13 C NMR spectra, while two carbon signals of C 5 H 4 appear upfield and one carbon signal of C 5 H 4 appears downfield compared to the carbon signals of C 5 H 5 .