Electrochemical Characterization of a Novel Organoelectrocatalyst, 7-Azabicyclo[2.2.1]heptan-7-ol (ABHOL), and Its Application to Electrochemical Sensors

Abstract

Electrochemical enzyme sensors are suitable for simple monitoring methods, for example, as glucose sensors for diabetic patients; however, they have several disadvantages arising from the properties of the enzyme. Therefore, non-enzymatic electrochemical sensors using functional molecules are being developed. In this paper, we report the electrochemical characterization of a new hydroxylamine compound, 7-azabicyclo[2.2.1]heptan-7-ol (ABHOL), and its application to glucose sensing. Although the cyclic voltammogram for the first cycle was unstable, it was reproducible after the second cycle, enabling electrochemical analysis of ethanol and glucose. In the first cycle, ABHOL caused complex reactions, including electrochemical oxidation and comproportionation with the generated oxoammonium ions. The electrochemical probe performance of ABHOL was more efficient than the typical nitroxyl radical compound, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and had similar efficiency to 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO), which is activated by the bicyclic structure. The results demonstrated the advantages of ABHOL, which can be synthesized from inexpensive materials via simple methods.

Introduction

Electrochemical measurement is a simple, easy, and rapid assay method used for various medical and environmental measurements.^1,2) In particular, diabetic glucose sensors based on the enzymatic reaction of glucose oxidase are used to measure blood glucose levels from the current generated by the electrolysis of hydrogen peroxide produced by glucose oxidase in the blood sample.^3,4) Enzymatic sensors provide high temporal resolution and substrate specificity because they rely on enzymatic reactions, although it is difficult to fabricate a sensor for a molecule that is not involved in an enzymatic reaction. In addition, the enzyme properties mean that enzymatic sensors have drawbacks such as long-term stability, price, and lot-to-lot reproducibility. Therefore, nonenzymatic electrochemical sensors have been developed using various functional materials including metal oxides⁵⁾ and sugar-responsive materials, such as phenylboronic acid.^6–8) We have focused on nitroxyl radical compounds, which can work as organocatalysts, to develop electrochemical sensors.^9–13)

Nitroxyl radical compounds, such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), are used in combination with appropriate oxidants, such as NaClO, to catalyze the oxidation of alcohols in organic synthesis.^14,15) Alcohol can also be oxidized electrochemically by applying an electric potential instead of by oxidants.^16,17) The response current obtained is proportional to the alcohol concentration in the measurement solution, thereby allowing alcohols or compounds with hydroxy groups in the molecule to be quantified.¹⁸⁾ However, TEMPO, a typical nitroxyl radical compound, requires the four methyl groups adjacent to its nitroxyl radical moiety to stabilize the radical, resulting in substantial steric hindrance around the active site and low reactivity. 9-Azabicyclo[3.3.1]nonane N-oxyl (ABNO)¹⁹⁾ and 2-azaadamantane N-oxyl (AZADO)^20–22) have been developed to solve this problem by stabilizing the radicals with bicyclic and tricyclic structures, respectively. These compounds have reduced steric hindrance and high reactivity. We have also developed 3-hydroxy-8-azabicyclo[3.2.1]octane N-oxyl (3-HO-ABOO) (previously named nortropine N-oxyl (NNO), whereas here a more systematic name is used for clarity), and reported electrochemical sensing of glucose and drugs under physiological conditions based on these compounds.^9–13)

We reported that 7-azabicyclo[2.2.1]heptan-7-ol (ABHOL), a highly active hydroxylamine catalyst with a new structure, exhibits high catalytic activity for alcohol oxidation under aerobic oxidation conditions using a copper salt as a co-catalyst.²³⁾ ABHOL is oxidized to the corresponding nitroxyl radical (7-azabicyclo[2.2.1]heptane N-oxyl; ABHO) under oxidation reaction conditions and exhibits oxidation catalytic activity. ABHOL was synthesized in six steps from trans-4-aminocyclohexanol (32.00 USD/25 g from TCI America), which is cheaper than nortropine (103.00 USD/25 g from TCI America), the starting material for 3-HO-ABOO. In 2008, Onomura and colleagues reported the electrochemical oxidation of alcohols catalyzed by a mixture of ABHO and ABHOL.²⁴⁾ However, the electrochemical analytical properties of ABHOL were unknown because ABHOL was intended for synthetic organic chemistry applications and has a long reaction time. In this study, the electro-oxidation properties of purified ABHOL were evaluated by cyclic voltammetry (CV) and its properties as an electrochemical analytical probe were compared with those of TEMPO and ABNO.

Experimental

Materials

ABNO¹⁹⁾ and ABHOL²³⁾ were synthesized according to the literature. TEMPO was purchased from TCI. All other reagents were special grade and were used without purification. The structures of the organocatalysts used in this study are shown in Fig. 1.

Fig. 1. Chemical Structures of the Organocatalysts Used in This Study

Electrochemical Measurement

CV was performed in a three-electrode cell consisting of a glassy carbon electrode (GC, diameter: 3 mm) as the working electrode, a Pt wire as the counter electrode and an Ag/AgCl reference electrode using an electrochemical analyzer (ALS model 660B, BAS, Tokyo, Japan). The surface of GC disk electrode was polished with alumina slurry (1 µm) and sonicated twice in distilled water for 5 min. CV cycles were recorded in 100 mM phosphate-buffered solution (10 mL, pH 7.0 or 7.4) at a scan rate of 100 mV/s. Multiple measurements were taken and a calibration curve was prepared using the average of the measurements. All experiments were performed at room temperature (approx. 20 °C).

Results and Discussion

We compared the electrochemical behavior of ABHOL and ABNO based on CV (Fig. 2). Cyclic voltammograms of ABNO showed reversible electrochemical responses with oxidation and reduction peaks around + 540 and + 470 mV vs. Ag/AgCl, respectively. The same reversible voltammogram was obtained for three cycle sweeps. Nitroxyl radicals were oxidized to oxoammonium ions by sweeping to higher potentials and the oxoammonium ions were reduced to the nitroxyl radicals by sweeping to lower potentials. Similar electrochemical responses were also observed in TEMPO.^16,17)

Fig. 2. Cyclic Voltammograms of 1 mM (a) ABHOL and (b) ABNO in 100 mM Phosphate Buffer (pH 7.0), Sweep Rate 100 mV/s

The cyclic voltammogram of ABHOL showed different and more complex behavior compared with ABNO. In the first cycle, a large oxidation peak and no reduction peak was observed. This unique CV shape is rationalized if the following complex reactions occurred: since a nitroxyl radical was not present in the solution, the proton-coupled electron transfer reaction from ABHOL (hydroxylamine) to the corresponding nitroxyl radical and the electron transfer reaction from the nitroxyl radical to the corresponding oxoammonium ion occurred at the same potential, as did the comproportionation reaction of the generated oxoammonium ion and the abundant ABHOL to the nitroxyl radical. In the second cycle, since some amounts of the nitroxyl radical were present around the working electrode, the electron transfer of the nitroxyl radical occurred at a lower potential than the proton-coupled electron transfer of the hydroxylamine, so the current value increased at a lower potential and the peak current value was lower than in the first cycle. No reduction peak was observed after the second cycle due to the comproportionation reaction.

To examine the suitability of ABHOL as an electrochemical probe, the electrolytic oxidation capacity of ABHOL was evaluated by CV in the presence of ethanol (Fig. 3). A calibration curve was obtained from the increase in peak oxidation current (ΔIp) in the first and second cycle of the cyclic voltammogram by measuring the CV of 1 mM ABHOL in the presence of 0 to 100 mM ethanol (Fig. 3 inset). In the first cycle, the apparent increase in ΔIp was small because the background current was higher due to several reactions proceeding simultaneously. By contrast, the increase in ΔIp in the second cycle was concentration-dependent for ethanol, and a well-calibrated curve was obtained. The oxoammonium ionic form of ABHOL produced by electrode oxidation was reduced by ethanol in solution and oxidized by the electrode again. ΔIp increased with ethanol concentration as this reaction proceeded repeatedly during the sweep. Even in neutral aqueous solution, an ethanol-concentration-dependent increase in oxidation current was observed at a sweep rate of 100 mV/s. The peak oxidation potential in 100 mM ethanol was about 15 µA, which was sufficient for use as a probe for electrochemical sensing. In our previous experiment, when 100 mM ethanol was electrolyzed with AZADO under similar conditions (pH 7.4, 100 mV/s), ΔIp of 5.5 µA was obtained.¹¹⁾ ABHOL had better performance as an electrochemical probe than AZADO because of its more compact bicyclic structure, which reduces steric hindrance around the nitroxyl radical moiety.²³⁾ In addition, the high oxidation potential may have contributed to the high peak current. The proposed electrochemical reaction of ABHOL based on these results is shown in Chart 1.

Fig. 3. Cyclic Voltammograms of 1 mM ABHOL in the Presence of Ethanol (0, 0.1, 0.3, 1, 3, 10, 30, 100 mM)

(a) First and (b) second cycle in 100 mM phosphate buffer (pH 7.0), sweep rate 100 mV/s. (Inset) Calibration curve of current ΔIp for ethanol concentration obtained from CV. The average values of 3 measurements with standard deviation are plotted.

Chart 1. Chart of the Electrochemical Reaction of ABHOL

ABHOL can be detected glucose using the same principle as ethanol by electrochemical oxidation of the hydroxyl group.⁹⁾ Figure 4a shows the second cycle of the cyclic voltammogram of ABHOL in the presence of 0 to 100 mM glucose, in which a concentration-dependent increase in glucose response was observed. Figure 4b shows the calibration curves for glucose obtained with ABHOL, ABNO, and TEMPO via similar measurements. The TEMPO and ABNO curves were plotted using ΔIp obtained from the first cycle of CV, whereas the ABHOL curve was plotted using ΔIp obtained from the first and second cycle of CV. For ABHOL, sensing with improved reproducibility was achieved by using ΔIp obtained from the second CV cycle rather than the first. The electrochemical response (linearity and range of calibration curves) of ABHOL to glucose was similar to that of ABNO. TEMPO could not be used to determine glucose concentration under these conditions.

Fig. 4. Glucose Response of Nitroxyl Radical Compounds

(a) Second cycle of cyclic voltammograms of 1 mM ABHOL in the presence of glucose (0, 0.5, 1, 2, 4, 6, 8, 10 mM) in 100 mM phosphate buffer (pH 7.4), sweep rate 100 mV/s. (b) Calibration curve for glucose obtained from ΔIp of ABHOL, ABNO, and TEMPO. The average values of 3 measurements with standard deviation are plotted. The regression equation is a plot of ABHOL (2nd cycle).

ABNO is synthesized from acetonedicarboxylic acid, glutaraldehyde, and aqueous ammonia in a three-step process, which involves a complicated Wolff-Kishner reduction and requires advanced synthetic organic chemistry techniques. In contrast, although ABHOL requires a six-step synthesis, the starting material, trans-4-aminoyclohexanol, is relatively cheap, and several of the steps are simple reactions, such as protection-deprotection, and have high yields, making ABHOL suitable for use as a general-purpose analytical probe.²³⁾

Conclusion

ABHOL was characterized as an electrochemical analysis probe. The electrolytic oxidation capacity of ABHOL was higher than that of TEMPO and similar to that of ABNO. ABHOL can be synthesized in high yield from low-cost materials. ABHOL is stable in the solid state at 4 °C for at least one year. Therefore, ABHOL is useful for the development of non-enzymatic electrochemical sensors. However, this method has no substrate specificity because it detects compounds with hydroxyl groups. In addition, the sensitivity is not enough for use in clinical applications. High-performance nitroxyl radical compounds that improve on these problems are expected to be developed.

Acknowledgments

This study was supported in part by the Takeda Science Foundation (K.S.), and JSPS KAKENHI Grant Nos. 23H02598 and 21H05210 (Digitalization-driven Trans-formative Organic Synthesis (Digi-TOS)) (Y.S.).

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Hernández-Rodríguez J. F., Rojas D., Escarpa A., Anal. Chem., 93, 167–183 (2021).
• 2) Jing L., Xie C., Li Q., Yang M., Li S., Li H., Xia F., Anal. Chem., 94, 269–296 (2022).
• 3) Soldà A., Valenti G., Marcaccio M., Giorgio M., Pelicci P. G., Paolucci F., Rapino S., ACS Sens, 2, 1310–1318 (2017).
• 4) Nascimento R. A. S., Özel R. E., Mak W. H., Mulato M., Singaram B., Pourmand N., Nano Lett., 16, 1194–1200 (2016).
• 5) Wei M., Qiao Y., Zhao H., Liang J., Li T., Luo Y., Lu S., Shi X., Lu W., Sun X., Chem. Commun., 56, 14553–14569 (2020).
• 6) Li J., Bai Z., Mao Y., Sun Q., Ning X., Zheng J., Electroanalysis, 29, 2307–2315 (2017).
• 7) Takahashi S., Anzai J., Langmuir, 21, 5102–5107 (2005).
• 8) Yang M., Ma C., Ding S., Zhu Y., Shi G., Zhu A., Anal. Chem., 91, 14029–14035 (2019).
• 9) Sato K., Ono T., Yoshida K., Ito T., Kashiwagi Y., Electroanalysis, 27, 2272–2274 (2015).
• 10) Ono T., Sugiyama K., Komatsu S., Kumano M., Yoshida K., Dairaku T., Fujimura T., Sasano Y., Iwabuchi Y., Kashiwagi Y., Sato K., RSC Advance, 11, 21622–21628 (2021).
• 11) Kashiwagi Y., Ono T., Sato F., Kumano M., Yoshida K., Dairaku T., Sasano Y., Iwabuchi Y., Sato K., Sens. Biosens. Res., 27, 100302 (2020).
• 12) Sugiyama K., Sasano Y., Komatsu S., Yoshida K., Ono T., Fujimura T., Iwabuchi Y., Kashiwagi Y., Sato K., Chem. Pharm. Bull., 69, 1005–1009 (2021).
• 13) Kumano M., Sugiyama K., Sato F., Komatsu S., Watanabe K., Ono T., Yoshida K., Sasano Y., Iwabuchi Y., Fujimura T., Kashiwagi Y., Sato K., Anal. Sci., 39, 369–374 (2023).
• 14) Tebben L., Studer A., Angew. Chem. Int. Ed., 50, 5034–5068 (2011).
• 15) Vogler T., Studer A., Synthesis, 2008, 1979–1993 (2008).
• 16) Semmelhack M. F., Schmid C. R., Cortes D. A., Tetrahedron Lett., 27, 1119–1122 (1986).
• 17) Semmelhack M. F., Chou C. S., Cortes D. A., J. Am. Chem. Soc., 105, 4492–4494 (1983).
• 18) Yamauchi Y., Maeda H., Ohomori H., Chem. Pharm. Bull., 44, 1021–1025 (1996).
• 19) Shibuya M., Tomizawa M., Sasano Y., Iwabuchi Y., J. Org. Chem., 74, 4619–4622 (2009).
• 20) Iwabuchi Y., Chem. Pharm. Bull., 61, 1197–1213 (2013).
• 21) Shibuya M., Sasano Y., Tomizawa M., Hamada T., Kozawa M., Nagahama N., Iwabuchi Y., Synthesis, 2011, 3418–3425 (2011).
• 22) Shibuya M., Tomizawa M., Suzuki I., Iwabuchi Y., J. Am. Chem. Soc., 128, 8412–8413 (2006).
• 23) Toda M., Sasano Y., Takahashi M., Kasabata K., Sato K., Kashiwagi Y., Iwabuchi Y., J. Org. Chem., 88, 1434–1444 (2023).
• 24) Demizu Y., Shiigi H., Oda T., Matsumura Y., Onomura O., Tetrahedron Lett., 49, 48–52 (2008).