3.2. Synthesis of Eight Types of GPx Mimics
The synthesis route of the eight types of GPx mimics is shown in Scheme 1
The regiospecific monotosylation of the 2-substituent hydroxyl group of cyclodextrin was carried out according to Wang et al. [22
]. 0.96 g of NaOH was dissolved in 160 mL of water and 4 g of recrystallized β-CD was weighed. While stirring, the acetone solution of p-TsCl (4 g/10 mL) was slowly added dropwise and the pH of the solution was kept slowly at pH = 12.5. About 4 h drip finished, continue to react 1 h. The pH of the solution was adjusted to 7.0 with 1 M HCl, 200 mL of methanol was added and the insoluble material was removed by suction filtration. The solution was distilled off to about 50 mL under reduced pressure, 50 mL of methanol was added, and the mixture was allowed to stand at 4 °C for one week and the precipitate was filtered off. Sephadex G-25 column chromatography, H2
O as eluent, collecting the first peak. The powder after lyophilization is 2-OTs-β-CD.
The regiospecific monotosylation of the 6-substituent hydroxyl group of cyclodextrin was carried out according to Matsui et al. [23
]. 6.0 g of NaOH was dissolved in 200 mL of water, 10 g of recrystallized β-CD was added, ice bath and stirred. A solution of p-TsCl in acetone (3.36 g/10 mL) was slowly added and the reaction was carried out at about 0 °C for 5 h. The insoluble matter was removed by suction filtration and the pH of the solution was adjusted to 7.0 with 1 M HCl. The solution was allowed to stand in boiling water and the insoluble material was removed by suction filtration through a Buchner funnel and a suction filter bottle preheated at 100 °C. The filtrate was allowed to stand at room temperature and then cooled at 4 °C overnight. The purification step was repeated three times. The precipitate was finally collected and dried in vacuo at 60 °C in an oven.
NaHSe and Na2
were prepared according to Klayman et al. [24
]. 10 mL H2
O was added into the vials, 200 mg NaBH4
(synthesis of Na2
was 106 mg) and 110 mg Se powder (synthesis of Na2
was 220 mg) was added and covered the plug and continued to bubble through with pure nitrogen. Placed on a magnetic stirrer to stir the reaction until the selenium powder is completely reduced, the solution was colorless with a small amount of white precipitated (the solution of Na2
NaHTe and Na2
were prepared according to Anslyn et al. [25
]. 10 mL H2
O was added into the vial, bottle A, bubbled through with pure nitrogen gas for 15 min then sealed. Then another vial, bottle B, was made by adding 160 mg Te (synthesis of Na2
was 84 mg) and 300 mg NaBH4
(synthesis of Na2
was 330 mg), bubbled through with pure nitrogen gas for 15 min, 10 mL H2
O solution was removed with a syringe into bottle B. Bottle B was then placed into the heating stirrer at 60 °C water bath stirring, the reaction process continued through the nitrogen. Tellurium powder reaction completely sealed, the solution was colorless (the solution of Na2
was deep purple).
The synthetic process was carried out according to Breslow and Liu et al. [26
]. 2-OTs-β-CD or 6-OTs-β-CD 330 mg was dissolved in 10 mL H2
O (two drops of DMF was added to 6-OTs-β-CD reaction mixture). The reaction mixture was then bubbled through with pure nitrogen gas for 20 min, and then 1 mL NaHSe/NaHTe (synthesis of dual-bridged mimics was Na2
) was added. Next the mixture was bubbled through with pure nitrogen gas for 15 min then sealed and stirred for 48 h (synthesis of dual-bridged mimics was 72 h) at 60 °C. The reaction solution was removed and centrifuged at 5000 rpm for 20 min. The precipitate was discarded and twice the volume of absolute ethanol was added. Was centrifuged at 5000 rpm for 20 min, collected precipitation. Was dissolved in 5 mL H2
O, separated by Sephadex G-25 column chromatography, and H2
O was used as eluent. The first peak was collected and lyophilizated.
We used 13C-NMR, ESI-MS, and elemental analysis to characterize the newly synthesized mimics.
2-SediCD. The 2-SediCD was obtained in 32.01% yield as a white with a little yellow powder. 13C-NMR (D2O): δ 103.3, 101.3, 82.1, 80.1, 73.4, 72.9, 69.8, 60.4; MALDI-MS: Calcd 2366.9, Found 2367.2; Anal. Calcd for C84H138O69Se·2H2O: C 43.58, H 6.01. Found: C 43.44, H 6.12.
2-SeCD. The 2-SeCD was obtained in 40.60% yield as a light yellow powder. 13C-NMR (D2O): δ 103.2, 101.8, 82.7, 80.3, 73.9, 72.8, 70.5, 61.9; MALDI-MS: Calcd 2517.7, Found 2518.2; Anal. Calcd for C84H138O69Se2·6H2O: C 40.32, H 5.96. Found: C 40.14, H 6.00.
6-SediCD. The 6-SediCD was obtained in 31.85% yield as a white with a little yellow powder. 13C-NMR (D2O): δ 102.3, 82.1, 73.5, 72.8, 72.3, 61.7, 59.3; MALDI-MS: Calcd 2366.9, Found 2367.4; Anal. Calcd for C84H138O69Se·2H2O: C 43.45, H 6.13. Found: C 43.58, H 6.07.
6-SeCD. The 6-SeCD was obtained in 39.21% yield as a light yellow powder. 13C-NMR (D2O): δ 103.7, 81.6, 73.2, 72.5, 71.8, 63.5, 59.8; MALDI-MS: Calcd 2518.0, Found 2516.9; Anal. Calcd for C84H138O69Se2·6H2O: C 40.32, H 5.99. Found: C 40.38, H 5.87.
2-TediCD. The 2-TediCD was obtained in 25.37% yield as a yellow powder. 13C-NMR (D2O): δ 104.3, 102.2, 82.5, 79.8, 73.5, 72.3, 69.9, 61.3; MALDI-MS: Calcd 2381.6, Found 2382.2; Anal. Calcd for C84H138O68Te·H2O: C 42.69, H 5.88. Found: C 42.62, H 5.95.
2-TeCD. The 2-TeCD was obtained in 33.41% yield as an orange powder. 13C-NMR (500 MHz, D2O), δ 101.8, 98.5, 80.1, 75.9, 72.3, 70.9, 68.3, 59.5. MALDI-MS: Calcd 2599.2, Found 2598.3; Anal. Calcd for C84H138O68Te2·6H2O: C, 38.32; H, 5.58. Found: C, 37.87, H, 5.65.
6-TediCD. The 6-TediCD was obtained in 28.48% yield as a yellow powder. 13C NMR (500 MHz, D2O): δ 102.6, 81.3, 72.9, 72.1, 71.8, 60.2; MALDI-MS: Calcd 2579.7, found 2579.9; Anal. Calcd for C84H138O68Te·12H2O: C, 39.07; H, 6.28. Found: C, 38.93; H, 6.05.
6-TeCD. The 6-TeCD was obtained in 33.41% yield as an orange powder. 13C NMR (500 MHz, D2O): δ 102.4, 81.5, 73.6, 72.5, 72.3, 60.5; MALDI-MS: Calcd 2689.4 Found 2688.3; Anal. Calcd for C84H138O68Te2·11H2O: C, 36.91; H, 5.95. Found: C, 36.75; H, 5.68.
The enzyme activity was measured at different pH (4–12) and at different temperatures, and the results show that the optimum pH and optimum temperature are 2-SediCD (pH 8.0, 55.3 °C), 2-SeCD (pH 7.8, 52.5 °C), 6-SediCD (pH 8.2, 58.2 °C), 6-SeCD (pH 7.9, 54.1 °C), 2-TediCD (pH 8.2, 52.3 °C), 2-TeCD (pH 7.9, 49.6 °C), 6-TediCD (pH 8.3, 54.6 °C), 6-TeCD (pH 8.1, 52.8 °C), respectively.