Synthesis of Ce6
We developed two new methods, i.e., method 1 and method 2, which included three steps: a) extraction of chlorophyll a from spirulina powder, b) demetallation of chlorophyll a to convert it into pheophytin a, and c) conversion to Ce6 from pheophytin a. These methods were accomplished by altering the reaction procedure as well as different conditions with each other (Scheme 2).
Method 1. Chlorophyll a extraction and Synthesis of Ce 6
a) Extraction of chlorophyll a
Spirulina (200 g) was dispersed in EtOH (2L) and stirred overnight (12 h) in a continuous nitrogen environment. The spirulina was filtered and the filtered ethanol solution was evaporated through a vacuum evaporator until the volume of ethanol remained to 400 mL. Then, water (200 mL) and hexane (500 mL) were added to the chlorophyll a containing EtOH solution, stirred for 10 min and kept in the refrigerator overnight. Later, chlorophyll a was extracted in hexane by liquid-liquid layer separation three times.
b) Conversion to pheophytin a
To the hexane solution of chlorophyll a, 1N HCl (7 mL) was added dropwise and stirred for 4 h at room temperature. 1M NaOH (10 mL) was added dropwise after the completion of the reaction analyzed by TLC. Then, 70% EtOH (400 mL) was added to the reaction mixture, which was again kept in the refrigerator for 12 h. The hexane layer was separated by liquid-liquid separation with 70% EtOH three times, vacuum evaporated, and then dried to obtain pheophytin a.
c) Conversion and purification of Ce6
The pheophytin a (4.92 g) was dissolved in acetone (400 mL) and nitrogen bubbled into the solution of pheophytin a for 30 min. 1M NaOH (20 mL) was added dropwise and refluxed overnight under inert conditions. After completion of the reaction monitored by TLC, the reaction mixture (Ce6) was filtered and washed with acetone. The black solid of Ce6 was collected and dried for 4 h. The dried Ce6 (3.92 g) was dissolved in 25 mL of water and stirred for 2 h at room temperature under inert conditions. 1N HCl (7.5 mL) was added to the solution to maintain pH 7.0 and stirred for 2 h. The reaction mixture was centrifuged at 10,000 rpm for 1.5 h to remove the junk like carbohydrates, peptides, and others. The supernatant was filtered, and then the filtrate was treated with 1N HCl to maintain pH ~ 3.5 and the Ce6 was collected through filtration. It was vacuum dried at 35 oC for 4 h to get 1.75 g yield (1% of spirulina powder). The Ce6 was analyzed through NMR and LC/MS. 1H-NMR (600 MHz, DMSO-d6); δ 9.75 (s, 1H), 9.65 (s, 1H), 9.10 (s, 1H), 8.23 (dd, 1H, J = 18, 18 Hz), 6.41 (dd, 1H, J = 18, 18 Hz), 6.13 (dd, 1H, J = 18, 18 Hz), 5.37–5.41 (q, 2H, J = 18 Hz), 4.61–4.65 (q, 1H, J = 6 Hz), 4.45–4.47 (q, 1H, J = 6 Hz), 3.71–3.75 (q, 2H, J = 6, 12 Hz), 3.59 (s, 3H), 3.49 (s, 3H), 3.25 (s, 3H), 2.63–2.68 (m, 1H), 2.29–2.34 (m, 1H), 2.14–2.19 (m, 1H), 1.68 (d, 6H, J = 6 Hz), 1.65 (t, 1H, J = 6, 12 Hz) Mass spectra m/z C34H36N4O6 (M + H)+: 597. Yield: 1.75 g, < 1% of spirulina powder
Method 2. Chlorophyll a extraction and Synthesis of Ce 6
a) Extraction of chlorophyll a
Spirulina (100 g) was dispersed in hexane (200 mL) for 30 min, 96% EtOH (800 mL) was added, and stirred overnight (12 h) in a continuous flow of nitrogen. Then, spirulina was filtered, water (200 mL) was added to the filtrate, and the hexane layer was separated through the liquid-liquid separation method to obtain chlorophyll a.
b) Conversion to pheophytin a
To the hexane solution of chlorophyll a, 5 mL of 2N HCl was added dropwise and stirred for 2 h. After completion of the reaction checked by TLC, the reaction mixture was washed through liquid-liquid separation by using 80% EtOH (200 mL) twice. The pheophytin a in the hexane layer was collected, vacuum evaporated, and dried for an hour.
c) Conversion and purification of Ce6
The pheophytin a (2.5 g) was dissolved in acetone (60 mL) and nitrogen bubbled into the solution of pheophytin a for 30 min. Then, 13% KOH in ethanol (378 mL) was added dropwise and refluxed for 10 min. After the completion of the reaction checked by TLC, the reaction mixture was cooled down to 17 oC and kept on an ice-bath. 11% v/v HCl was added to the reaction mixture to maintain pH 0.2 and stirred for an hour for acidification. Then 30% NaOH solution was added to the solution to maintain pH 0.6 and stirred for 30 min for Ce6 cleaning while maintaining a temperature of 17–19 oC. The reaction solution was filtered where almost all the junk like carbohydrates, peptides, carotenoids, and others was removed. The filtered solution was again treated with the addition of 30% NaOH solution to maintain the pH ~ 3.0 and stirred for an hour. Thus, the formed precipitate of Ce6 was filtered and dried for 24 h at 25 oC. The synthesized Ce6 was analyzed through NMR and LC/MS. 1H-NMR (600 MHz, DMSO-d6); δ 9.73 (s, 1H), 9.61 (s, 1H), 9.10 (s, 1H), 8.21 (dd, 1H, J = 18 Hz, 18 Hz), 6.39 (dd, 1H, J = 18), 6.12 (dd, 1H, J = 18, 18 Hz), 5.37–5.39 (q, 2H, J = 7.2 Hz), 4.61–4.65 (q, 1H, J = 6 Hz), 4.45–4.47 (q, 1H, J = 12 Hz), 3.71–3.74 (q, 2H, J = 6 Hz), 3.59 (s, 3H), 3.48 (s, 3H), 3.22 (s, 3H), 2.63–2.69 (m, 1H), 2.30–2.34 (m, 1H), 2.14–2.19 (m, 1H), 1.68 (d, 6H, J = 6 Hz), 1.63–1.66 (t, 1H, J = 6, 12 Hz) Mass spectra m/z C34H36N4O6 (M + H)+: 597. Yield: 0.8 g, < 1% of spirulina powder.