Antioxidant Activity of Planar Catechin Conjugated with Trolox

1. Introduction

Oxidative stress is associated with the onset of and progression of various diseases such as cancer, diabetes, neurodegenerative diseases (Alzheimer’s disease, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis), arteriosclerosis, and heart disease [1,2,3,4]. The mitochondria, the main source of reactive oxygen species (ROS), contribute to oxidative stress due to electron leakage in the electron transport chain during energy production [5]. Mitochondrial damage exacerbates this process, leading to the production of even more ROS. Excessive ROS production results in cell damage or cell death. Furthermore, ROS generated by inflamed cells or wounds cause oxidative damage to lipids and proteins in the cell membrane, as well as to enzymes and DNA, leading to various diseases [6]. Therefore, consuming substances that can eliminate ROS and reduce oxidative stress is effective in preventing the onset and progression of diseases associated with oxidative stress [7]. Various natural substances can eliminate ROS and suppress oxidative stress [8,9]. The consumption of red wine, known for its effectiveness in preventing coronary artery disease, has been shown to be due to the antioxidant effects of polyphenol compounds, such as resveratrol, catechin, epicatechin, and anthocyanin [10,11]. Among these, catechin is found not only in grapes but also green tea and apples. Catechin, with its catechol structure, reduces and eliminates ROS. It also has a chelating effect on metals, helping to eliminate metals involved in ROS generation [12]. In fact, catechin intake has been reported to have a preventative effect against diseases caused by oxidative stress [13].

Given that (+)-catechin has a structure where the chroman skeleton (AC ring) and catechol skeleton (B ring) are perpendicular to each other, we introduced an isopropyl group at the 3-position of the AC ring and the C6’-position of the C ring to synthesize a planar catechin (PCat) with a fixed 3D structure on a plane [14]. PCat exhibits radical-scavenging activity five times stronger than (+)-catechin and has been shown to possess powerful α-glucosidase inhibition, amyloid β aggregation inhibition, antitumor, and antiviral effects [15,16].

Tocopherol, a representative fat-soluble antioxidant, suppresses the oxidation of polyunsaturated fatty acids, abundant in biological membranes, into lipid peroxides through radical chain reactions [17]. Tocopherol eliminates free radicals generated during the lipid peroxidation process through reducing actions. Although tocopherol itself is oxidized in this process, vitamin C can reduce tocopherol, allowing it to regain its antioxidant effect [18]. The oxidized vitamin C is then regenerated by glutathione, NADH, and other substances, forming an antioxidant network in the body [19]. This defense mechanism effectively counters many oxidative stresses [20,21,22]. We hypothesized that if a single molecule could have multiple structures with radical-scavenging activity, an antioxidant network could form between these structures, exerting an even more powerful antioxidant effect.

In the present research, we aimed to form oxidative networks within a molecule to exert a more potent antioxidant effect. To achieve this, we synthesized a compound, PCat–TrOH, where PCat was conjugated with Trolox (TrOH), a water-soluble analog of α-tocopherol, and demonstrated its radical-scavenging activity (Figure 1).

2. Materials and Methods

2.1. General Methods

Unless otherwise noted, all commercially available reagents (Wako Chemicals, Tokyo, Japan; Tokyo Chemical Industry, Tokyo, Japan; Sigma-Aldrich, St. Louis, MO, USA) were used as received without purification. Solvents for chromatography were of industrial grade, and the process of all reactions were monitored by thin-layer chromatography that was on silica gel 60 F254 (0.25 nm, Merck). Column chromatography was performed on silica gel 60 (spherical, 63-210 μm, KANTO CHEMICAL). ¹H and ¹³C NMR spectra were recorded with a JEOL JNMAL-400 (400 MHz) NMR spectrometer using CDCl₃, DMSO-d6 as the solvent and tetramethylsilane as the internal reference. Mass spectra were measured using a JMS-700V (JEOL) mass spectrometer. The purity of all compounds was determined to be >95% using ¹H and ¹³C NMR spectroscopy.

2.2. Synthesis of PCat-TrOH

2.2.1. Ethyl 3-((6aS,12aR)-2,3,8,10-tetrahydroxy-5-methyl-5,6a,7,12a-tetrahydroisochromeno [4,3-b]chromen-5-yl)propanoate (1)

Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 5.42 mL, 30.0 mmol) was slowly added to a solution of (+)-catechin (6.0 g, 20.7 mmol) and ethyl 4-oxovalerate (7.14 mL 51.7 mmol) in dry tetrahydrofuran (THF) (200 mL) at –30 °C. After stirring for 20 h, the mixture was poured into water and extracted using diethyl ether (3 × 150 mL). The organic layer was washed with brine, dried over Na₂SO₄, and filtered. The solvent was evaporated, and the residue was purified using silica gel chromatography (4:1:0.1 toluene/acetone/methanol) to give 6.89 g (80.0%) of 1 as a white powder: m.p. 170 – 173 ^oC; ¹H-NMR (400 MHz, DMSO-d₆) δ 9.30 (s, 1H), 9.11 (s, 1H), 9.06 (s, 1H), 8.86 (s, 1H), 6.93 (s, 1H), 6.50 (s, 1H), 5.94 (d, J = 2.0Hz, 1H), 5.78 (d, J = 2.0Hz, 1H), 4.38 (d, J = 9.2Hz, 1H), 3.95 (q, J = 6.8Hz, 2H), 3.72 (m, 1H), 2.78 (dd, J = 5.6 and 15.2Hz, 1H), 2.28 (dd, J = 10.4 and 15.2Hz, 1H), 2.01 (t, J = 7.6Hz, 2H), 1.83 (t, J = 7.6Hz, 2H), 1.45 (s, 3H), 1.12 (t, J = 7.2Hz 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.0, 156.7, 156.8, 155.2, 145.1, 144.4, 131.3, 124.7, 111.9, 111.4, 99.1, 95.6, 94.2, 76.1, 72.3, 65.9, 59.7, 28.7, 27.5, 27.2, 26.6, 14.1; HRMS (ESI+) m/z [M⁺] calcd for C₂₂H₂₄O₈, 416.1471, found 416.1475.

2.2.2. Ethyl 3-((6aS,12aR)-2,3,8,10-tetrakis((tert-butyldimethylsilyl)oxy)-5-methyl-5,6a,7,12a-tetrahydroisochromeno[4,3-b]chromen-5-yl)propanoate (2)

To a solution of 1 (5.18 g, 12.4 mmol) in N,N-dimethylformamide (100 mL), tert-butyldimethylchlorosilane (11.25g, 74.6 mmol) and imidazole (5.08 g, 74.6 mmol) were added at 0°C under argon. After stirring at room temperature for 20 h, the mixture was diluted with ethyl acetate, washed with brine, dried over sodium sulfate (Na₂SO₄). The solvent was evaporated and the residue was subjected to silica gel column chromatography (1:3, n-hexane/ethyl acetate) to give 8.44g (78% yield) of 2 as a white solid: m.p. 104-106 ^oC;; ¹H-NMR (400 MHz, CDCl₃) δ7.07 (s, 1H), 6.57 (s, 1H), 6.42 (d, J = 2.0Hz, 1H), 6.29 (d, J = 2.2Hz, 1H), 4.55 (d, J = 8.8Hz 1H), 4.04 (q, J = 7.2Hz, 2H), 3.88 (m, 1H), 3.13 (m, 1H), 2.59 (m, 1H), 2.11 (m, 2H), 1.82 (m, 2H), 1.49 (s, 3H), 1.18 (t, J = 7.2Hz, 3H), 1.02 (s, 18H), 0.99 (s, 9H), 0.98 (s, 9H), 0.24 (s, 12H), 0.19(s, 6H), 0.180 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.1, 157.3, 155.4, 154.4, 146.4, 145.3, 131.6, 127.5, 120.2, 119.7, 108.7, 103.8, 97.5, 85.2, 80.6, 73.5, 62.6, 37.8, 32.9, 31.3, 31.1, 26.7, 26.5, 25.1, 14.1, -0.8, -1.1; HRMS (ESI+) m/z [M⁺] calcd for C₄₆H₈₀O₈Si₄, 872.4930, found 872.4938.

2.2.3. 3-((6aS,12aR)-2,3,8,10-tetrakis((tert-butyldimethylsilyl)oxy)-5-methyl-5,6a,7,12a-tetrahydroisochromeno[4,3-b]chromen-5-yl)propan-1-ol (3)

A solution of 2 (6.81 g, 7.8 mmol) in dry THF (100 mL) was added dropwise to a solution of 1M lithium aluminum hydride (LiAlH₄) in THF (11.7 mL, 11.7 mmol) and dry THF (20 mL) at 0 ^oC and then stirred for 20 h at room temperature. The mixture was cooled on ice, and saturated potassium sodium tartrate was added dropwise; subsequently, the mixture was filtered through a small pad of Celite and thoroughly washed with ethyl acetate. The organic solution was washed with brine, dried over Na₂SO₄, and filtered. The solvent was evaporated, and the residue was purified using silica gel chromatography (1:6 n-hexane/ethyl acetate) to give 3.24 g (50%) of 3 as a white powder: m.p. 144–147 ^oC; ¹H-NMR (400 MHz, CDCl₃) δ 7.10 (s, 1H), 6.48 (s, 1H), 6.09 (d, J = 2.2 Hz, 1H), 5.97 (d, J = 2.2 Hz, 1H), 4.51 (d, J = 9.6 Hz, 1H), 3.87 (m, 1H), 3.55 (m, 2H), 3.02 (dd, J = 6.0 and 15.6Hz, 1H), 2.51 (dd, J = 10.8 and 15.6 Hz, 1H), 1.91 (m, 2H), 1.53 (s, 3H), 1.28 (m, 2H), 1.01 (s, 18H), 0.99 (s, 9H), 0.98 (s, 9H), 0.29 (s, 12H), 0.19 (s, 6H), 0.18 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 157.8, 153.3, 152.0, 141.6, 140.4, 129.4, 126.9, 118.5, 115.1, 107.3, 104.6, 96.1, 85.0, 84.2, 76.1, 60.5, 39.6, 33.3, 31.3, 31.1, 27.3, 26.7, 26.5, 24.4, -0.8, -1.1; HRMS (ESI+) m/z [M + H⁺] calcd for C₄₄H₇₉O₇Si₄, 831.4904, found 831.4910.

2.2.4. tert-butyl (tert-butoxycarbonyl)(3-((6aS,12aR)-2,3,8,10-tetrakis((tert-butyldimethylsilyl)oxy)-5-methyl-5,6a,7,12a-tetrahydroisochromeno[4,3-b]chromen-5-yl)propyl)carbamate (4)

To a stirred solution of 3 (5.99 g, 7.2 mmol), di-tert-butyl iminodicarboxylate (6.26g, 28.8 mmol), and triphenylphosphine (7.57 g, 28.8 mmol) in 100 mL of dry THF, 40 % diethyl azodicarboxylate in toluene (13.11 ml, 28.8 mmol) was slowly added under argon at 0 °C. After stirring at room temperature for 20 hours, the mixture was diluted with ethyl acetate, washed with brine and dried over Na₂SO₄. The solvent was evaporated and the residue was subjected to silica gel column chromatography (1:10, n-hexane/ethyl acetate) to give 5.57 g (75 % yield) of 4 as a white solid: m.p. 67–72 ^oC; ¹H NMR (400 MHz, CDCl₃) δ 7.08 (s, 1H), 6.46 (s, 1H), 6.10 (d, J = 2.2 Hz, 1H), 5.97 (d, J = 2.2 Hz, 1H), 4.61 (d, J = 9.2 Hz, 1H), 3.80 (m, 1H), 3.49 (m, 2H), 3.01 (dd, J = 6.0 and 15.8 Hz, 1H), 2.47 (dd, J = 10.4 and 15.8 Hz, 1H), 1.75 (m, 2H), 1.53 (m, 2H), 1.51 (s, 3H), 1.46 (s, 18H), 1.01 (s, 18H), 0.98 (s, 18), 0.25 (s, 12H), 0.18 (s, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 159.3, 156.6, 154.9, 150.5, 144.4, 140.3, 131.8, 127.6, 117.3, 115.6, 108.7, 106.1, 152.3, 83.9, 82.8, 80.5, 73.4, 44.6, 37.5, 33.0, 31.0, 30.9, 28.1, 27.3, 26.7, 26.5, 17.5, -0.8, -1.1; HRMS (ESI+) m/z [M + H⁺] calcd for C₅₄H₉₆NO₁₀Si₄, 1030.6112, found 1030.6121.

2.2.5. N-(3-((6aS,12aR)-2,3-bis((tert-butyldimethylsilyl)oxy)-8,10-dihydroxy-5-methyl-5,6a,7,12a-tetrahydroisochromeno[4,3-b]chromen-5-yl)propyl)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide (5)

To a solution of 4 (2.10 g, 2.1 mmol) in dichloromethane (200 mL), trifluoroacetic acid (20 mL) was slowly added at 0 °C under argon. After stirring at room temperature for 2.5 hours, the solvent was evaporated. The residue containing the amine was dried in vacuum and then used in the next reaction. To a solution of TrOH (0.48 g, 1.9 mmol) in N,N-dimethylformamide (DMF: 20 mL) was added N,N-diisopropylethylamine (1.1 mL, 6.4 mmol) followed by 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-Oxide Hexafluorophosphate (HATU: 1.61 g, 4.2 mmol). The mixture was stirred at room temperature for 15 min, after which the time the dark clear mixture was treated with the solution of the amine and N,N-diisopropylethylamine (1.1 mL, 6.4 mmol) in DMF (20 mL). After stirring at room temperature for 24 hours, the mixture was diluted with ethyl acetate, washed with brine, dried over Na₂SO₄ and the solvent was evaporated. The residue was purified by silica gel column chromatography (1:1, n-hexane/ethyl acetate) to give 0.67 g (30 % yield) of 5 as a white solid: m.p. 167–170 ^oC; ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (s, 1H), 9.05 (s, 1H), 7.47 (s, 1H), 7.11 (m, 1H), 7.01 (s, 1H), 6.51 (s, 1H), 5.95 (d, J = 2.2 Hz, 1H), 5.80 (d, J = 2.2 Hz, 1H), 4.41 (d, J = 8.4 Hz, 1H), 3.74 (m, 1H), 2.98 (m, 2H), 2.83 (m, 1H), 2.42 (m, 2H), 2.30 (m, 1H), 2.11 (m, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H), 1.71 (m, 2H), 1.55 (m, 2H), 0.98 (s, 9H), 0.94 (s, 9H), 0.21 (s, 6H), 0.18 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.8, 157.7, 156.8, 156.0, 147.3, 146.8, 146.0, 145.7, 132.7, 129.3, 128.0, 121.5, 118.5, 117.7, 116.3, 114.0, 97.4, 96.3, 94.2, 92.4, 81.9, 80.8, 76.5, 42.5, 41.0, 36.2, 33.3, 31.1, 30.0, 28.3, 26.9, 22.3, 21.0, 16.8, 12.5, 11.8, -0.8; HRMS (ESI+) m/z [M + H⁺] calcd for C₄₆H₆₈NO₉Si₂, 834.4433, found 834.4439.

2.2.6. 6-hydroxy-2,5,7,8-tetramethyl-N-(3-((6aS,12aR)-2,3,8,10-tetrahydroxy-5-methyl-5,6a,7,12a-tetrahydroisochromeno[4,3-b]chromen-5-yl)propyl)chromane-2-carboxamide (PCat–TrOH)

To a solution of 5 (0.76 g, 0.72 mmol) in dry THF (200 mL), 1M tetrabutylammonium fluoride in THF(3.93 ml, 3.93 mmol) was slowly added at 0 °C under argon. After stirring at room temperature for 1.5 hours, the mixture was diluted with ethyl acetate, washed with brine, dried over Na₂SO₄. The solvent was evaporated, and the residue was purified by column chromatography (2:1:0.1, toluene/acetone/methanol to give 0.22g (50% yield) of PCat–TrOH as a white solid: m.p. 186–189 ^oC; ¹H NMR (400 MHz, DMSO-d₆) δ 9.29(s, 1H), 9.05 (s, 2H), 8.74(s, 1H), 7.48 (s, 1H), 7.16 (m, 1H), 6.90 (s, 1H), 6.38 (s, 1H), 5.94 (s, 1H), 5.78 (s, 1H), 4.34 (m, 1H), 3.69 (m, 1H), 2.95 (m, 2H), 2.80 (m, 1H), 2.42 (m, 2H), 2.31 (dd, J = 10.4 and 14.8 Hz, 1H), 2.14 (m, 2H), 2.09 (s, 3H), 2.07 (s, 3H), 1.99 (s, 3H), 1.68 (m, 2H), 1.51 (m, 2H), 1.34 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.6, 157.8, 156.6, 155.6, 146.5, 148.8, 145.0, 144.0, 132.7, 129.0, 127.5, 121.0, 117.5, 116.0, 114.1, 113.0, 98.0, 96.8, 94.8, 92.4, 81.0, 79.2, 74.5, 42.0, 40.2, 36.0, 34.4, 32.4, 26.9, 22.3, 21.2, 16.8, 12.6, 11.5; HRMS (ESI+) m/z [M + H⁺] calcd for C₃₄H₄₀NO₉, 606.2704, found 606.2709.

2.3. Antioxidant activity measurements

Galvinoxyl radical (GO^•) was commercially obtained from Tokyo Chemical Industry Co., Ltd., (Tokyo, Japan), and GO^• is the highest available purity and was used without further purification unless otherwise noted. Acetonitrile (MeCN) used as solvent was commercially obtained from Nacalai Tesque Inc. (Kyoto, Japan) and used as received. The rates of GO^•-scavenging reactions using test samples in MeCN were determined by monitoring the absorbance change at 428 nm due to GO^• (ε = 1.4 x 10⁵ M^-1 cm^-1) after mixing it in MeCN at a volumetric rate of 1:1 using a stopped-flow technique on a UNISOKU REP-1000-02 NM spectrophotometer (UNISOKU Co., Ltd., Osaka, Japan) at 298 K. The pseudo-first-order rate constants (k_obs) were determined by a least-squares curve fitting using an UNITCOM SOLUTION-M06M-134-UHX (UNIT.COM INC., Osaka, Japan). The first-order plots of ln(A − A_∞) vs. time (A and A_∞ were denoted as the absorbance at the reaction time and the final absorbance, respectively) were linear with the correlation coefficient ρ > 0.999 until three or more half-lives. In all cases, the k_obs values obtained from at least three independent measurements agreed within ±5% experimental error. In all cases, solutions were normally equilibrated with air.

2.4. Spectral titrations

Typically, a 5 μL of PCat–TrOH (2.63×10^-4 M) in MeCN was added to GO^• (7.77 x 10^-6 M) in MeCN, and the absorbance at 428 nm due to GO^• was plotted with respect to the concentration ratio, [PCat–TrOH]/[GO^•]. The UV–vis spectral changes accompanying the reaction were monitored and measured using an Agilent 8453 photodiode array spectrophotometer thermostated with a Peltier temperature control at 298 K (Agilent Technologies, Santa Clara, CA, USA). In all cases, solutions were normally equilibrated with air.

3. Results

3.1. Chemistry

We designed and synthesized a compound, PCat–TrOH, in which TrOH was conjugated to PCat, creating a molecule with multiple radical-scavenging structures (Figure 2). The PCat site, where the 3D catechin structure was fixed, was obtained through the Oxa-Pictet Spengler reaction between (+)-catechin and ethyl levulinate. Specifically, 1 was synthesized by treating a tetrahydrofuran (THF) solution of catechin and ethyl levulinate with trimethylsilyl trifluoromethanesulfonate (TMSOTf). Next, the four hydroxyl groups were protected with tert-butyldimethylchlorosilyl (TBS) groups, after which the ester structure was reduced with lithium aluminum hydride (LiAlH₄) to obtain alcohol (3). Then, 3 underwent a Mitsunobu reaction using (Boc)₂NH as a pronucleophile in the presence of Ph₃P and DEAD to obtain 4. Trifluoroacetic acid (TFA) was used to remove the BOC group together with the TBS groups on A ring, after which the amino group of 4 underwent a condensation reaction with the carboxyl group of TrOH using the condensing agent DIPEA/HATU, without purification, to obtain compound 5. Finally, TBAF was used for deprotection, resulting in PCAT–TrOH.

2.4. Radical Scavenging Activity

The radical-scavenging activities of PCat–TrOH, TrOH, PCat, and Cat were evaluated in acetonitrile using the galvinoxyl radical (GO^•), a model compound for ROS. The strong absorption of GO^• at 428 nm immediately disappeared upon reaction with the antioxidant. Therefore, the change in color of the GO^• solution in the presence of the antioxidant was used to measure the radical-scavenging activity rates of PCat–TrOH, TrOH, PCat, and Cat. This spectral change was monitored by measuring the decrease in absorbance at 428 nm using the stopped-flow method (Figure 3). The concentrations of PCat–TrOH, TrOH, PCat, and Cat were set to over 10 times the concentration of GO^• to measure the decrease in absorbance at 428 nm under pseudo-first-order reaction conditions, allowing us to obtain the pseudo-first order rate constant (k_obs). The pseudo-first-order rate constant (k_obs) increased linearly with increasing concentrations of PCat–TrOH, TrOH, PCat, and Cat (Figure 4). The slopes of the linear plots of k_obs vs PCat–TrOH, TrOH, and PCat showed that the second-order rate constants (k) of the radical-scavenging activity were 9.6 × 10 ² M⁻¹ s⁻¹, 1.1 × 10³ M⁻¹ s⁻¹, and 1.7 × 10 ² M⁻¹ s ⁻¹, respectively. We have also previously reported that the k of Cat under the same conditions was 2.6 × 10 ¹ M⁻¹ s ⁻¹. The k value of PCat was approximately 6.5 times that of Cat, indicating that the planar structure increased its radical-scavenging activity. Meanwhile, TrOH was even more powerful than PCat, with a k value approximately 6.5 times greater than that of Cat. The k value of the newly synthesized compound PCat–TrOH, which binds TrOH to PCat, was approximately 5.6 times larger than that of planar catechin, exhibiting an almost equally powerful radical-scavenging activity as that of TrOH alone.

2.4. Spectral titrations

ROS are eliminated by one-electron reduction, so catechin eliminates two ROS and is oxidized to quinone. Additionally, α-tocopherol reduces two lipid peroxides and is itself reduced to tocopheryl quinone. Therefore, we sought to clarify the number of GO^• radicals that could be eliminated by PCat–TrOH (antioxidant capacity) through titration (Figure 5), which we conducted by adding antioxidants to a GO^• solution, plotting the reduced 428 nm absorbance of GO^•, and finally obtaining the molar ratio of antioxidants to GO^• when GO^• was completely eliminated. As shown in Figure 6, in the spectral titration conducted in this study, the 428 nm absorbance of GO^• was completely eliminated by 0.25 times the quantity of PCat–TrOH relative to GO^•, indicating that the molar ratio of PCat–TrOH to GO^• was 1:4. Meanwhile, the 428 nm absorbance was eliminated by adding 0.5 times the quantity of PCat or TrOH relative to GO^•, indicating that their molar ratios were 1:2. Therefore, it was thought that PCat–TrOH eliminated four GO^• radicals, compared to PCat or TrOH, which eliminated only two GO^• radicals.